HK1168114A - Variant immunoglobulins with improved manufacturability - Google Patents

Description

Variant immunoglobulins with improved producibility

The present invention relates to the expression of antibody molecules and, in particular, to the production of antibody molecules with improved producibility.

The term "aggregation" describes a variety of phenomena, all of which involve self-association of proteins, can occur in a variety of environments, and can occur from cell culture and fermentation to isolation, purification, and preparation processes. For example, "aggregation" is often used when describing the following: forming inclusion bodies; protein accumulation in the "insoluble" fraction after cell fractionation; turbidity, protein precipitation or particle formation in the sample; or the formation of small soluble oligomers, etc. Protein aggregation is herein understood to be "abnormal self-association" of polypeptide chains and may have a variety of causes and manifestations, depending primarily on the nature of the molecules involved and the environment in which the polypeptides are produced, isolated, stored and administered.

The three-dimensional structure of a polypeptide determines its natural physiological function. Proteins are usually synthesized in ribosomes to obtain their native structure. The processing of protein folding is fundamentally encoded in the amino acid sequence of the protein. However, a variety of molecular aids and quality control mechanisms that ensure that the polypeptide acquires its intended biologically active structure can also assist in folding in vivo, thereby avoiding populations of abnormal conformations in the process. These external aids and quality control elements include chaperones, disulfide isomerases, post-translational modifications (i.e., proteolytic processes, acylation, glycosylation, etc.), and ubiquitin-proteasome systems.

In addition, protein aggregation is driven by the intrinsic stability of the protein in solution. Proteins are only poorly stable in solution, and this relatively low stability is important in conferring specific biological properties while providing good regulatory mechanisms to control their action. However, for example, the departure of a polypeptide from its natural environment for the development of a drug poses a number of severe limitations in the production, isolation and formulation of the polypeptide, which can have a dramatic impact on the stability and aggregation behavior of the polypeptide.

Changes to the natural environment of a given protein, particularly due to specific production or process requirements, can have a dramatic effect on the stability of the protein and in solution. This problem is exacerbated by process requirements to increase yield, such as: artificial synthesis, heterologous expression (i.e. prokaryotic expression of mammalian proteins), use of secretory or non-secretory pathways, saturation of protein synthesis structure; or other obstacles caused by non-native proteins such as polypeptide fragments or fusions. For example, expression of mammalian proteins in heterologous systems poses a number of risks. This is due to the fact that the system generally lacks a suitable molecular environment to ensure "natural" folding and processing.

The present invention relates to the following findings: modification of certain key positions in the amino acid sequence of an immunoglobulin molecule leads to productive improvements and in particular to a reduction in aggregation propensity and/or an increase in production levels.

One aspect of the invention provides a method of producing a variant immunoglobulin comprising:

providing a parent immunoglobulin which is capable of producing,

introducing a substitution in the VL domain framework region and/or the aggregation prone segment of VL CDR3 of the parent immunoglobulin; and/or the presence of a gas in the gas,

introducing substitutions in the VH framework region and/or aggregation prone segments of VH CDR 3; and/or

Introducing a substitution in an aggregation-prone segment of the CH1 constant region domain of the parent immunoglobulin heavy Chain (CH),

thereby producing a variant immunoglobulin.

The variant immunoglobulin may have improved producibility relative to the parent immunoglobulin. For example, the variant immunoglobulin may exhibit a reduced propensity for aggregation and/or increased production capacity when expressed relative to the parent immunoglobulin.

For example, a variant immunoglobulin may exhibit at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 200%, or at least 500% increase in production capacity relative to the parent immunoglobulin, and/or a variant immunoglobulin may exhibit at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 90%, or at least 99% decrease in aggregation (i.e., decrease in the proportion of molecules in the assembly in the native state that undergo aggregation) relative to the parent immunoglobulin. Variant immunoglobulins may exhibit up to 100% reduction in aggregation (i.e., complete elimination of aggregation) relative to the parent immunoglobulin.

The improvement in producibility may result, in whole or in part, from a reduced tendency to aggregate relative to the parent immunoglobulin.

Aggregation propensity relates to the propensity of immunoglobulins to form insoluble aggregates upon expression in recombinant systems. The reduced propensity for aggregation reduces the proportion of molecules in the immunoglobulin's native state ensemble that are present in aggregated form (Carpenter et al, 2009J Pharm Sci. Apr; 98 (4): 1201-5). In other words, the proportion of molecules in aggregated or insoluble form in the natural state ensemble of the variant immunoglobulin is lower than the proportion of molecules in aggregated or insoluble form in the natural state ensemble of the parent immunoglobulin.

Variant immunoglobulins may exhibit less self-association or aggregation than the parent immunoglobulin under native conditions or at increased temperatures (e.g., 60 ℃), i.e., conditions under which the antigen binding site of the immunoglobulin does not develop. Preferably, the variant immunoglobulin exhibits less self-association or aggregation than the parent immunoglobulin under native conditions (e.g., conditions that do not result in unfolding of the immunoglobulin).

The aggregation propensity described herein is distinct from the Thermal Refolding Efficiency (TRE), which relates to the ability of a Protein to refold correctly after thermal denaturation, and is typically determined using Circular Dichroism (CD) (Tanha et al Protein Eng Des Sel.2006 Nov; 19 (11): 503-9). The reduction in the aggregation propensity of the immunoglobulins described herein may have little or no effect on the thermal refolding efficiency of the immunoglobulins. Thus, thermal refolding efficiency is independent of aggregation propensity and has no significant impact on immunoglobulin producibility.

Aggregation can be determined by conventional methods. Suitable techniques include GP-HPLC, HPLC and AUC (Gabrielson JP et al J Pharm Sci 200796 (2): 268-79), protein loss after filtration, turbidity, fluorochrome binding (e.g.niler red, thioflavin T or 8-anilino-1-naphthalenesulfonic acid; see, e.g.Hawe, A.et al Pharmaceutical Research 200825(7)1487-99 or Demeule, B et al 2007 Int J Pharm 329: 37-45), field flow separation (FFF; Demeule, Bet al. mAbs 20091 (2): 142- -150) and analytical ultracentrifugation (AU/AUC; Liu J et al. AAPS J20068: 580-9).

Other suitable Methods are described in Arvinte T.In "Methods for structural analysis of proteins pharmaceuticals" AAPS Press, 2005: 661-6 and Kiese S et al J pharmSci 200897 (10): 4347-66.

The improvement in producibility may result, in whole or in part, from an increased capacity for production relative to the parent immunoglobulin.

Variant immunoglobulins may exhibit higher production capacities than the parent immunoglobulin. For example, variant immunoglobulins may exhibit increased yields or titers compared to the parent immunoglobulin when expressed in recombinant systems such as bacterial or mammalian cells. Productivity can be measured using standard techniques, such as Bradford assay, spectrophotometry, and ELISA.

Variant immunoglobulins may also exhibit one or more of the following properties relative to the parent immunoglobulin: improved purification yield, reduced configuration difficulty, reduced immunogenicity, and increased bioavailability.

In preferred embodiments, the improvement in producibility may result from both a reduced tendency to aggregate and an increased production capacity relative to the parent immunoglobulin.

Preferably, the variant immunoglobulin exhibits the same or substantially the same activity (i.e., antigen binding activity) as the parent immunoglobulin.

An immunoglobulin is a polypeptide or protein that contains an antigen binding site. An antigen binding site is a portion of an immunoglobulin that specifically binds to and is spatially complementary to part or all of an antigen. When the antigen is large, the immunoglobulin may bind only a certain portion of the antigen, which portion is called an epitope. Preferably, the antigen binding domain comprises an immunoglobulin light chain variable region (VL) and an immunoglobulin heavy chain variable region (VH), which may be on the same or different polypeptide chains.

Examples of immunoglobulins include whole antibodies, including antibody isotypes such as IgG, IgA, IgD, IgM, and IgE and their isotypic subclasses such as IgG1 and IgG 4; an antibody fragment; and engineered antibody derivatives such as Small Immunoproteins (SIP), miniaturised antibodies, camelid VHH domains and diabodies.

Examples of antibody fragments include (i) Fab fragments consisting of the VL, VH, CL and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) (ii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) dAb fragments (Ward, E.S.et al, Nature 341, 544-546(1989)) consisting of VH or VLDomain composition; (v) an isolated CDR region; (vi) f (ab')₂A fragment which is a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules (scFv) in which the VH domain and the VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-; (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", which are multivalent or multispecific fragments constructed by gene fusion (WO 94/13804; P. Holliger et al, Proc. Natl. Acad. Sci. USA 906444-6448, 1993). Fv, scFv or diabody molecules can be stabilized by introducing a disulfide bond linking the VH and VL domains (Y.Reiter et al. Nature Biotech 141239-. Minibodies comprising scFv linked to the CH3 domain can also be prepared (S.Hu et al, Cancer Res.5638-30611996).

Immunoglobulins may be natural or wholly or partially synthetic. Thus, chimeric immunoglobulins comprising an antigen binding domain or equivalent fused to another polypeptide may also be included. Cloning and expression of chimeric antibodies is described in EP-A-0120694 and EP-A-0125023. Various artificial immunoglobulins comprising one or more antigen binding sites have been engineered, including for example Fab₂、Fab₃Diabodies, triabodies, tetrabodies and minibodies.

In some preferred embodiments, the parent and variant immunoglobulins described herein are whole antibodies or antibody fragments comprising VH and VL domains.

Immunoglobulins may be isolated or obtained by purification from natural sources, or may also be obtained by genetic recombination or chemical synthesis as described herein. For example, synthetic immunoglobulins can be produced by expression of genes produced by means of oligonucleotides synthesized and assembled in suitable expression vectors, as described by Knappik et al J.mol.biol. (2000)296, 57-86 or Krebs et al journal of Immunological Methods 254200167-84.

The immunoglobulin may be naturally glycosylated, or glycosylated in vitro or ex vivo, e.g., using a heterologous eukaryotic cell system (e.g., CHO cells), or the immunoglobulin may be unglycosylated (e.g., when produced by expression in prokaryotic cells). Preferably, the glycosylated immunoglobulin does not comprise fucose.

Suitable parent immunoglobulins may be any immunoglobulin having a known amino acid sequence and which may be produced by recombinant expression in a heterologous expression system. Heterologous expression systems include mammalian cells such as CHO cells, insect cells, yeast cells and bacterial cells (e.g., e.coli, particularly for antibody fragments), and are described in more detail below.

Parent immunoglobulins suitable for modification as described herein may exhibit suboptimal producibility. In other words, the parent immunoglobulin may exhibit one or more undesirable production traits, such as increased aggregation and/or decreased productivity relative to a control immunoglobulin when recombinantly expressed in a heterologous system at production scale.

Suitable control immunoglobulins include the same type of immunoglobulin which exhibit satisfactory producibility in the same heterologous system. When expressed on a production scale in mammalian systems, suitable parent immunoglobulins may, for example, yield soluble species of 1g/L or less (as determined by ELISA), and/or aggregation of 5% or more (as determined by GP-HPLC). When expressed in bacterial systems on a production scale, the parent immunoglobulin may, for example, yield soluble material of 0.5g/L or less (as determined by ELISA), and/or aggregation of 5% or more (as determined by GP-HPLC).

The methods may comprise determining the producibility of a parent immunoglobulin, for example by determining the level of production and propensity for aggregation as described herein. Parent immunoglobulins that produce low yields (e.g., 1g/L or less of soluble species in mammalian systems) and/or high aggregation tendencies (e.g., 5% or greater aggregation) can be identified as exhibiting suboptimal producibility.

Modifications of the parent immunoglobulin sequences described herein may be used to increase or optimize their producibility.

In a preferred embodiment, the parent immunoglobulin may be a therapeutic antibody, i.e., an antibody that binds to a therapeutically relevant antigen to achieve a beneficial therapeutic effect. Many examples of therapeutic antibodies are known in the art.

Variant immunoglobulins are non-naturally occurring immunoglobulins that bind the same target antigen as the parent immunoglobulin but have a different amino acid sequence. For example, the sequence of a variant immunoglobulin may differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues relative to the sequence of the parent immunoglobulin. Preferably, the sequence of the variant immunoglobulin differs from the parent immunoglobulin sequence only in one or more substitutions in the aggregation-prone segment, as described herein. In other words, the sequence of the variant immunoglobulin is preferably identical to the amino acid sequence of the parent immunoglobulin except for one or more substitutions as described herein.

The variant immunoglobulin binds to the same epitope as the parent immunoglobulin and competes with the parent immunoglobulin for binding to the antigen. Competition between immunoglobulins can be readily determined in vitro, for example, by ELISA and/or by adding specific reporter tags to one immunoglobulin so that it can be detected in the presence of other unlabelled immunoglobulins. When the parent immunoglobulin is a therapeutic antibody, the variant immunoglobulin preferably achieves the same beneficial therapeutic effect.

The antigen binding domain of the immunoglobulins described herein preferably comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). Each of the immunoglobulin VH and VL domains comprises 3 CDRs (CDR1, CDR2, and CDR3) separated by framework regions (FR1, FR2, FR3, and FR 4). CDRs are highly variable regions in the variable domain that contain a large proportion of the amino acid residues responsible for specific binding of an antibody to the antigen or epitope that it recognizes. The length of a CDR can be 2-26 amino acids, depending on the specific basic framework can accommodate the length.

Preferably, the immunoglobulins used as described herein lack intra-or inter-CDR disulfide bonds.

Parent immunoglobulin CH1, VH and VL domains suitable for use as described herein may be obtained from any germline or rearranged human variable domain, or may be synthetic variable domains based on consensus sequences of known human variable domains. In some embodiments, the VH and VL domains of the variant immunoglobulin produced may lack one or more CDR sequences (e.g., CDR3), e.g., for use in antibody engineering as described below.

Variant immunoglobulins may exhibit improved producibility, e.g., reduced aggregation and/or increased production capacity relative to a parent immunoglobulin, when recombinantly expressed in a heterologous system on a production scale. When expressed on a production scale in a mammalian system, the variant immunoglobulin may, for example, yield a soluble substance yield of greater than 1g/L (as determined by ELISA), and/or less than 5% aggregation (as determined by GP-HPLC). Preferably, the variant immunoglobulins described herein simultaneously produce a soluble material yield of greater than 1g/L and exhibit less than 5% aggregation. When expressed on a production scale in a bacterial system, the variant immunoglobulin may, for example, yield a soluble substance yield of more than 0.5g/L (determined by ELISA), and/or less than 5% aggregation (determined by GP-HPLC). Preferably, the variant immunoglobulins described herein simultaneously produce a soluble mass yield of greater than 0.5g/L and exhibit less than 5% aggregation.

Aggregation-prone segments are short sequences of amino acid residues in the light or heavy chain sequences of an immunoglobulin that have a high aggregation propensity relative to surrounding sequences and adversely affect the producibility of the immunoglobulin. Aggregation-prone segments may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.

The aggregation-prone segment may, for example, be located in the CH1, VH or VL domain of an immunoglobulin. A single domain or region of an immunoglobulin, for example the FR1, FR2, FR3, FR4 or CDR3 regions from a VH or VL domain or a CH1 domain, may have multiple aggregation-prone segments within its sequence. Substitution of one or more residues in one or more of these aggregation-prone segments described herein may improve the producibility of the immunoglobulins described herein.

The positions of the immunoglobulin residues described below are numbered according to the scheme shown in the following data: kabat, e.a., Wu, t.t., Perry, h.m., Gottesmann, K.S & Foeller, c. (1991), Sequences of Proteins of Immunological Interest, 5 th edition, NIH publication No. 91-3242, U.S. department of health and human services.

Where appropriate, the substitution positions may be described with respect to the Kabat numbered residues of the immunoglobulin sequence which are not variant.

Alternative antibody numbering schemes are described in Honegger, A and Pl ü ckthun A (2001). Another numbering scheme for immunoglobulin variable domains: an Automatic modeling and analysis tool.J.mol.biol 309, 657-670. Tables 1a and 1b show the correspondence between the honeyger and Kabat numbering schemes.

The amino acid substitution in the aggregation-prone segment described herein is preferably one of the 20 naturally occurring amino acids. Naturally occurring amino acids and their standard one-letter and three-letter abbreviations are well known in the art (Principles of Protein Structure G.Schulz & R.Schirmer, (1979) Springer-Verlag NY Inc USA).

Substitutions may be introduced in the aggregation-prone segments of the VL domain framework regions of the parent immunoglobulin. Suitable aggregation-prone segments of the framework regions of the VL domain may be selected from the group consisting of position 20 aggregation-prone segments, position 37 aggregation-prone segments, position 45 aggregation-prone segments and position 74 aggregation-prone segments.

The 20-position aggregation prone segment is located in the VL framework region 1 and extends from position 15 to position 23 of the VL domain.

The substitution in the aggregation prone segment at position 20 may occur at position 18 of the VL domain.

The amino acid residue at position 18 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 18 is substituted with R, S or V.

For example, the amino acid residue at position 18 in a parent immunoglobulin may be T. Variant immunoglobulins may comprise VL domains comprising T18R, T18S, or T18V substitutions.

The substitution in the aggregation prone segment at position 20 may occur at position 20 of the VL domain.

The amino acid residue at position 20 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 20 is substituted with K, R, S or V.

For example, the amino acid residue at position 20 in a parent immunoglobulin may be T. Variant immunoglobulins may comprise VL domains comprising T20K, T20R, T20S or T20V substitutions.

The 37 th aggregation propensity segment is located in the VL framework region 2 and extends from position 36 to position 38 of the VL domain.

The substitution in the aggregation prone segment at position 37 may occur at position 37.

The amino acid residue at position 37 in a parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 37 is substituted with Q or N.

For example, the amino acid residue at position 37 in a parent immunoglobulin can be L. A variant immunoglobulin may comprise a VL domain comprising a L37Q substitution.

The 45 th aggregation propensity segment is located in the VL framework region 2 and extends from position 42 to position 49 of the VL domain.

The substitution in the aggregation prone segment at position 45 may occur at position 45.

The amino acid residue at position 45 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 45 is substituted with E, K, R, Q or V.

For example, the amino acid residue at position 45 in a parent immunoglobulin may be T. The variant immunoglobulin may comprise a VL domain comprising a T45E, T45K, T45R, T45Q, or T45V substitution. The amino acid residue at position 45 in the parent immunoglobulin may be Q. The variant immunoglobulin may comprise a VL domain comprising a Q45E, Q45K, or Q45R substitution.

Substitution in the aggregation prone segment at position 45 may occur at position 46.

The amino acid residue at position 46 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 46 is substituted with L, Y, K, R, H, F or S.

For example, the amino acid residue at position 46 in a parent immunoglobulin may be T. Variant immunoglobulins may comprise a VL domain comprising a T46L or T46Y substitution. The amino acid residue at position 46 in the parent immunoglobulin may be L. The variant immunoglobulin may comprise a VL domain comprising L46K, L46R, L46H, L46F, or L46S substitutions.

The aggregation prone region at position 74 is located in the VL framework region 3 and extends from position 71 to position 77 of the VL domain.

Substitution in the aggregation prone segment at position 74 may occur at position 74.

The amino acid residue at position 74 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 74 is substituted with V.

For example, the amino acid residue at position 74 in a parent immunoglobulin may be T. A variant immunoglobulin may comprise a VL domain comprising a T74V substitution.

Substitutions may be introduced in the aggregation-prone segment in the VL CDR3 of the parent immunoglobulin. Suitable aggregation-prone segments may be selected from VL CDR 3N-terminal aggregation-prone segments and VLCDR 3C-terminal aggregation-prone segments.

The N-terminal aggregation-prone segment of the VL CDR3 extends from the non-variant residue C88 in the VL domain sequence to amino acid 8 at the terminus of residue C88C. For example, the VL CDR 3N-terminal aggregation prone segment may extend from position 88 to position 95a of the VL domain.

One or more of the amino acid residues at position 88 (C88), position 89 (C88+ 1; i.e., the terminal residue of residue C88C), position 90 (C88+2), position 91 (C88+3), position 92 (C88+4), position 93 (C88+5), position 94 (C88+6), position 95 (C88+7) and position 95a (C88+8) in the parent immunoglobulin may be substituted with a different amino acid residue in the variant immunoglobulin. The VL CDR3 position numbered relative to the non-variant Kabat residue C88 is shown in parentheses.

Substitutions in the N-terminal aggregation-prone segment of the VL CDR3 may occur at position 91 (C88+ 3).

The amino acid residue at position 91 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 91 is substituted with A, F, L, R, S or W. For example, the amino acid residue at position 91 in a parent immunoglobulin may be Y. The variant immunoglobulin may comprise a VL domain comprising a Y91A, Y91F, Y91L, Y91R, Y91S, or Y91W substitution.

Substitutions in the N-terminal aggregation-prone segment of the VL CDR3 may occur at position 93 (C88+ 5).

The amino acid residue at position 93 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 93 is substituted with D, F, L, Q, S, T, W or Y. For example, the amino acid residue at position 93 in the parent immunoglobulin may be P, and the variant immunoglobulin may comprise a VL domain comprising a P93D, P93F, P93L, P93Q, P93S, P93T, P93W, or P93Y substitution.

The VL CDR 3C-terminal aggregation prone segment extends from the N-terminal amino acid position 3 (i.e., positions F98-3 or 95) of the non-variant residue F98 in the VL domain sequence to position 99.

One or more of the amino acid residues at position 95 (F98-3), 96 (F98-2), 97 (F98-1), 98 or 99 in the parent immunoglobulin may be substituted with a different amino acid residue in the variant immunoglobulin.

Substitutions in the VL CDR 3C-terminal aggregation prone segment may occur at position 96 (F98-2). The VL CDR3 position numbered relative to the variant Kabat residue F98 is shown in parentheses.

The amino acid residue at position 96 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 96 is substituted with E, L, V or W.

For example, the amino acid residue at position 96 in the parent immunoglobulin may be R, and the variant immunoglobulin may comprise a VL domain comprising a R96E, R96L, R96V, or R96W substitution.

Substitutions may be introduced in aggregation prone segments in the VH domain framework regions of the parent immunoglobulin. Suitable aggregation-prone segments of VH domain framework regions may be selected from FR1 aggregation-prone segments, aggregation-prone segments at position 75, aggregation-prone segments at position 95, and aggregation-prone segments at position 102 (or CDR3 aggregation-prone segments).

The FR1 aggregation prone segment is located in VH framework region 1 and may, for example, extend from position 1 to position 21 of the VH domain sequence.

The VH FR1 aggregation prone segment may comprise an aggregation prone segment selected from positions 1 to 3, 4 to 6, 11 to 13, and 16 to 21.

The VH FR1 aggregation prone segment may extend from position 1 to position 3 of the VH domain sequence.

Substitution in the VH FR1 aggregation prone segment may occur at position 1.

The amino acid residue at position 1 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 1 is substituted with E, Q, D, A or V. For example, the amino acid residue at position 1 in the parent immunoglobulin may be E, and the variant immunoglobulin may comprise a VH domain comprising an E1Q, E1D, E1A, or E1V substitution; or the amino acid residue at position 1 in the parent immunoglobulin may be Q, and the variant immunoglobulin may comprise a VH domain comprising a Q1E, Q1D, Q1A, or Q1V substitution.

The VH FR1 aggregation prone segment may extend from position 4 to position 6 of the VH domain sequence.

Substitutions in the VH FR1 aggregation prone segment may occur at position 5 of the VH domain.

The amino acid residue at position 5 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 5 is substituted with V. For example, the amino acid residue at position 5 in a parent immunoglobulin may be L, and a variant immunoglobulin may comprise a VH domain comprising an L5V substitution.

The VH FR1 aggregation prone segment may extend from position 11 to position 13 of the VH domain sequence.

Substitutions in the VH FR1 aggregation prone segment may occur at position 12 of the VH domain.

The amino acid residue at position 12 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 12 is substituted with V. For example, the amino acid residue at position 12 in a parent immunoglobulin may be L, and a variant immunoglobulin may comprise a VH domain comprising a L12V substitution.

The VH FR1 aggregation prone segment may extend from position 16 to position 21 of the VH domain sequence.

Substitution in the VH FR1 aggregation prone segment may occur at position 17.

The amino acid residue at position 17 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 17 is substituted with R. For example, the amino acid residue at position 17 in a parent immunoglobulin may be G, and a variant immunoglobulin may comprise a VH domain comprising a G17R substitution.

Substitution in the VH FR1 aggregation prone segment may occur at position 19.

The amino acid residue at position 19 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 19 is substituted with T or V. For example, the amino acid residue at position 19 in a parent immunoglobulin may be L, and a variant immunoglobulin may comprise a VH domain comprising an L19T or L19V substitution.

Substitution in the VH FR1 aggregation prone segment may occur at position 20.

The amino acid residue at position 20 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 20 is substituted with A, K, S or T. For example, the amino acid residue at position 20 in the parent immunoglobulin may be R, and the variant immunoglobulin may comprise a VH domain comprising a R20A, R20K, R20S, or R20T substitution.

The 75 th aggregation propensity segment may extend from position 60 to position 85 of the VH domain.

The substitution in the 75 th aggregation prone segment may occur at position 61.

The amino acid residue at position 61 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. For example, residue 61 may be substituted with K, R, Q, E, D, N or A. Preferably, the residue at position 61 is substituted with R. For example, the amino acid residue at position 61 in a parent immunoglobulin may be P, and a variant immunoglobulin may comprise a VH domain comprising a P61R substitution.

The substitution in the 75 th aggregation prone segment may occur at position 85.

The amino acid residue at position 85 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. For example, residue 85 may be substituted with E, D or A. Preferably, the residue at position 85 is substituted with E. For example, the amino acid residue at position 85 in the parent immunoglobulin may be V, and the variant immunoglobulin may comprise a VH domain comprising a V85E, V85D, or V85A substitution.

The VH 95 th aggregation prone segment is located in the VH framework region 3 and may extend from position 91 to position 99 or 100.

One or more of the amino acid residues in VH domain 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100 of the parent immunoglobulin may be substituted with a different amino acid residue in the variant immunoglobulin.

Substitution in the VH 95 th aggregation prone segment may occur at position 94.

The amino acid residue at position 94 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. For example, residue 94 may be substituted with R, K or T. Preferably, the residue at position 94 is substituted with R. For example, the amino acid residue at position 94 in the parent immunoglobulin may be K and the VH domain that the variant immunoglobulin may comprise comprises a K94R substitution, or the amino acid residue at position 94 in the parent immunoglobulin may be H and the VH domain that the variant immunoglobulin may comprise comprises a H94R, H94K or H94T substitution.

Substitution in the VH 95 th aggregation prone segment may occur at position 95.

The amino acid residue at position 95 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. For example, residue 95 may be substituted with E or D. Preferably, the residue at position 95 is substituted with D. For example, the amino acid residue at position 95 in a parent immunoglobulin may be R, and a variant immunoglobulin may comprise a VH domain comprising a R95E or R95D substitution.

Substitution in the VH 95 th aggregation prone segment may occur at position 96.

The amino acid residue at position 96 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 96 is substituted with a. For example, the amino acid residue at position 96 in a parent immunoglobulin may be G, and a variant immunoglobulin may comprise a VH domain comprising a G96A substitution.

Substitution in the VH 95 th aggregation prone segment may occur at position 100.

The amino acid residue at position 100 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. The residue at position 100 may be substituted with F, G, L, M or P. Preferably, the residue at position 100 is substituted with F. For example, the amino acid residue at position 100 in the parent immunoglobulin may be V, and the variant immunoglobulin may comprise a VH domain comprising a V100F, V100G, V100L, V100M, or V100P substitution.

Substitutions may be introduced in the VH CDR3 aggregation prone segment of the parent immunoglobulin.

The VH CDR3 aggregation prone segment extends from position 100c (i.e., the two N-terminal amino acids of the non-variant residue D101 or D101-2 numbered relative to Kabat residue D101) to position 102 or 103.

One or more of the amino acid residues at positions D101-2 (two amino acids N-terminal to residue D101), D101-1 (one amino acid N-terminal to residue D101), 101, 102 and 103 in the VH domain of the parent immunoglobulin may be substituted with a different amino acid residue in the variant immunoglobulin.

Substitutions in the aggregation-prone segment of the VH CDR3 may occur at position D101-2 (two amino acids N-terminal to residue D101).

The amino acid residues at positions D101-2 in the parent immunoglobulin may be substituted for different amino acid residues in the variant immunoglobulin. Preferably, the residue at position D101-2 is substituted with D, E, F, G, P, S, T, W or Y. For example, the amino acid residue at position D101-2 in the parent immunoglobulin may be A, and the variant immunoglobulin may comprise a VH domain comprising an A (D101-2) D, A (D101-2) E, A (D101-2) F, A (D101-2) G, A (D101-2) P, A (D101-2) S, A (D101-2) T or A (D101-2) W or A (D101-2) Y substitution. In other words, substitutions from a to D, E, F, G, P, S, T, W or Y at the two amino acid positions N-terminal to D101, e.g. a100 c.

Substitutions in the aggregation-prone segment of VH CDR3 may occur at position D101-1 (one amino acid from the N-terminus of D101).

The amino acid residue at position D101-1 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position D101-1 is substituted with F, G, L, P or M. For example, the amino acid residue at position D101-1 in the parent immunoglobulin may be S, and the variant immunoglobulin may comprise a VH domain comprising an S (D101-1) F, S (D101-1) G, S (D101-1) L, S (D101-1) P or S (D101-1) M substitution. In some embodiments, residue S (D101-1) corresponds to S100D.

Substitution in the VH CDR3 aggregation prone segment may occur at position 102.

The amino acid residue at position 102 in a parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 102 is substituted with A, F, H, I, L, V or Y. For example, the amino acid residue at position 102 in the parent immunoglobulin may be P, and the variant immunoglobulin may comprise a VH domain comprising a P102A, P102F, P102H, P102I, P102L, P102Y, or P102V substitution, or the amino acid residue at position 102 in the parent immunoglobulin may be S, and the variant immunoglobulin may comprise a VH domain comprising a S102A, S102F, S102H, S102I, S102L, S102Y, or S102V substitution.

Substitution in the VH CDR3 aggregation prone segment may occur at position 103.

The amino acid residue at position 103 in the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, the residue at position 103 is substituted with I, L or V. For example, the amino acid residue at position 103 in the parent immunoglobulin may be W, and the variant immunoglobulin may comprise a VH domain comprising a W103I, W103L, or W103V substitution.

The CH1 aggregation prone segment extends from position 150 to position 156.

Substitution in the CH1 aggregation prone segment may occur at position 153.

The amino acid residue at position 153 of the parent immunoglobulin may be substituted for a different amino acid residue in a variant immunoglobulin. Preferably, residue 153 is substituted with V. For example, the amino acid residue at position 153 in a parent immunoglobulin may be S, and a variant immunoglobulin may comprise a CH1 domain comprising a S153V substitution.

A variant immunoglobulin may comprise up to 10 substitutions as described herein with respect to the parent immunoglobulin sequence. For example, a variant immunoglobulin may comprise up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.

Substitutions may be in the same aggregation-prone segment or in different aggregation-prone segments. For example, a variant immunoglobulin may comprise substitutions in 1, 2, 3, 4, or more of the different aggregation-prone segments described herein. Variant immunoglobulins may comprise substitutions in the VH domain, VL domain and/or CH1 regions.

Any combination of substitutions described herein may be employed. Examples of suitable combinations of substitutions are shown in tables 3a and 3 b.

In some embodiments, a variant immunoglobulin may comprise a substitution at 2 in the VL FR1 and/or FR2 domains; comprises 3 substitutions in the VL CDR3 domain; comprises 2 substitutions in the VL FR2 domain and 3 substitutions in the VL CDR3 domain; comprises 2 substitutions in the VL FR1 domain, 2 substitutions in the VL FR2 domain and 3 substitutions in the VL CDR3 domain; comprises a substitution at 2 in the VH FR1 domain; comprises 1 substitution in the VH FR1 domain and 1 substitution in the VH CDR3 domain; comprises 2 substitutions in the VHCDR3 domain; or comprises 1 substitution in the CH1 domain and 1 substitution in the VHFR1 domain.

The variant immunoglobulin may comprise 2 substitutions in the aggregation-prone segment at position 20. For example, a variant immunoglobulin may comprise a VL domain comprising a substitution at positions 18 and 20 as described above. For example, residues at positions 18 and 20 may be substituted with R. The VL domain may, for example, comprise T18R and T20R substitutions.

The variant immunoglobulin may comprise a substitution at the aggregation-prone segment at position 20 and a substitution at the aggregation-prone segment at position 74. For example, a variant immunoglobulin may comprise VL domains comprising substitutions at positions 18 and 74 as described above. For example, the residue at position 18 may be substituted with R and the residue at position 74 may be substituted with V. The VL domain may, for example, comprise T18R and T74V substitutions.

The variant immunoglobulin may comprise a substitution at position 45 of 2 in the aggregation prone segment. For example, a variant immunoglobulin may comprise VL domains comprising substitutions at positions 45 and 46 described above. For example, residue 45 may be substituted with K or R and residue 46 may be substituted with L. The VL domain may, for example, comprise T45K (or T45R) and T46L substitutions.

The variant immunoglobulin may comprise a substitution at position 20 comprising 2 and a substitution at position 45 comprising 2 of the aggregation-prone segment. For example, a variant immunoglobulin may comprise a VL domain comprising a substitution at positions 18, 20, 45, and 46 described above. For example, the residues at positions 18 and 20 may be substituted with R, the residue at position 45 may be substituted with K or R, and the residue at position 46 may be substituted with L. The VL domain may, for example, comprise T18R, T20R, T45K and T46L substitutions or T18R, T20R, T45R and T46L substitutions.

The variant immunoglobulin may comprise 2 substitutions in the VL CDR3N end aggregation prone segment. For example, the variant immunoglobulin may comprise a VL domain comprising a substitution at position 91 (C88+3), 93 (C88+5), and 96 (F98-2) as described above. For example, residue 91 may be substituted with W, residue 93 may be substituted with Q, and residue 96 may be substituted with V. The VL domain may, for example, comprise Y91W (C88+3), P93Q (C88+5) and R96V (F98-2) substitutions.

A variant immunoglobulin may comprise a substitution at position 45 of the aggregation prone segment of 2 and a substitution at position 2 of the VL CDR 3N-terminal aggregation prone segment. For example, the variant immunoglobulin may comprise a substituted VL domain at positions 45, 46, 91 (C88+3), 93 (C88+5), and 96 (F98-2) described above. For example, the residue at position 45 may be substituted with K or R, the residue at position 46 may be substituted with L, the residue at position 91 may be substituted with W, the residue at position 93 may be substituted with Q, and the residue at position 96 may be substituted with V. The VL domain may, for example, comprise T45K, T46L, Y91W (C88+3), P93Q (C88+5) and R96V (F98-2) substitutions, or T45R, T46L, Y91W (C88+3), P93Q (C88+5) and R96V (F98-2) substitutions.

A variant immunoglobulin may comprise a substitution at position 20 of the aggregation prone segment of 2, a substitution at position 45 of the aggregation prone segment of 2, and a substitution at 2 or 3 at the N-terminal aggregation prone segment of VL CDR 3. For example, a variant immunoglobulin may comprise a VL domain comprising a substitution at positions 18, 20, 45, 46, 91 (C88+3), 93 (C88+5), and 96 (F98-2) described above. For example, the residues at positions 18 and 20 may be substituted with R, the residue at position 45 may be substituted with K or R, the residue at position 46 may be substituted with L, the residue at position 91 may be substituted with W, the residue at position 93 may be substituted with Q, and the residue at position 96 may be substituted with V. The VL domain may, for example, comprise T18R, T20R, T45K, T46L, Y91W (C88+3), P93Q (C88+5) and R96V (F98-2) substitutions, or T18R, T20R, T45R, T46L, Y91W (C88+3), P93Q (C88+5) and R96V (F98-2) substitutions.

The variant immunoglobulin may comprise a VL domain comprising a substitution at VL 37 aggregation prone segment and VL 45 aggregation prone segment. For example, a variant immunoglobulin may comprise VL domains comprising substitutions at positions 37 and 45 described above. For example, the residue at position 37 may be substituted with Q or N, and the residue at position 45 may be substituted with E, K or R. The VL domain may, for example, comprise L37Q and Q45R substitutions.

The variant immunoglobulin may comprise a substituted VH domain in the VH FR1 aggregation prone segment and the VH CDR3 aggregation prone segment.

For example, a variant immunoglobulin may comprise a VH domain comprising a substitution at positions 1 and 102 as described above. For example, residue 1 may be substituted with Q, A, E or V and residue 102 may be substituted with Y. The VH domain may, for example, comprise Q1A and P102Y substitutions, Q1E and P102Y substitutions, Q1V and P102Y substitutions, E1A and P102Y substitutions, E1Q and P102Y substitutions or E1V and P102Y substitutions.

In other examples, the variant immunoglobulin may comprise a VH domain comprising a substitution at positions 1 and 103 described above. For example, residue 1 may be substituted with Q, A, D, E or V and residue 103 may be substituted with L. The VH domain may, for example, comprise Q1A and W103L substitutions, Q1E and W103L substitutions, Q1D and W103L substitutions, Q1V and W103L substitutions, E1A and W103L substitutions, E1Q and W103L substitutions, E1D and W103L substitutions, or E1V and W103L substitutions.

The variant immunoglobulin may comprise 2 substitutions in the VH CDR3 aggregation prone segment.

For example, a variant immunoglobulin may comprise a VH domain comprising a substitution at position 100c (D101-2) and 100D (D101-1) as described above. For example, the residue at position 100c may be substituted with W and the residue at position 100d may be substituted with F. The VH domain may, for example, comprise a100cW and S100dF substitutions.

In other examples, the variant immunoglobulin may comprise a VH domain comprising a substitution at position 100D (D101-1) and position 102 as described above. For example, residue 100d may be substituted with F or M and residue 102 may be substituted with V or Y. The VH domain may, for example, comprise S100dF and P102V substitutions, S100dM and P102V substitutions, S100dF and P102Y substitutions or S100dM and P102Y substitutions.

In other examples, the variant immunoglobulin may comprise a VH domain comprising a substitution at positions 102 and 103 described above. For example, residue 102 may be substituted with V or Y and residue 103 may be substituted with L, I or V. The VH domain may, for example, comprise P102Y and W103L substitutions, P102Y and W103I substitutions, P102Y and W103V substitutions, P102V and W103L substitutions, P102V and W103I substitutions or P102V and W103V substitutions.

The variant immunoglobulin may comprise an aggregation prone segment at position 95 of VH and the VH CDR3 aggregation prone segment comprises a substituted VH domain. For example, a variant immunoglobulin may comprise a VH domain comprising a substitution at positions 95 and 102 as described above. For example, residue 95 may be substituted with E or D and residue 102 may be substituted with A, F, H, I, L, V or Y. The VH domain may, for example, comprise R95D and S102V substitutions. Optionally, the VH domain may also comprise substitutions at positions 94 and 100 as described above. For example, residue 94 may be substituted with R, K or T and residue 100 may be substituted with F, G, L, M or P. The VH domain may, for example, comprise H94R, R95D, V100F and S102V substitutions.

The variant immunoglobulin may comprise a substitution in the CH1 aggregation prone segment and the VH 19 aggregation prone segment. For example, a variant immunoglobulin may comprise a CH1 domain comprising a substitution at position 153 as described above, and a VH domain comprising a substitution at position 19 as described above. For example, residue 153 may be substituted with V and residue 19 may be substituted with T. The immunoglobulin may, for example, comprise a S153V substitution in the CH1 domain and a L19T substitution in the VH domain.

In other examples, the variant immunoglobulin may comprise a CH1 domain comprising a substitution at position 153 as described above, and a VH domain comprising a substitution at position 20 as described above. For example, residue 153 may be substituted with V and residue 20 may be substituted with A. Variant immunoglobulins may, for example, comprise S153V and R20A substitutions.

A variant immunoglobulin may comprise a VL domain comprising a substitution in the VL 20 aggregation prone segment and a VH domain comprising a substitution in the VH FR1 aggregation prone segment. For example, a variant immunoglobulin may comprise a VH domain with a substitution at position 20, and a variant immunoglobulin may comprise a VL domain with a substitution at position 18. For example, the residue at position 20 in the VH domain may be substituted with A and the residue at position 18 in the VL domain may be substituted with V. For example, an immunoglobulin may comprise a VL domain having a T18V substitution, and a VH domain having a R20A or R20V substitution.

A variant immunoglobulin may comprise a VL domain comprising a substitution in the VL 45 aggregation prone segment and a VH domain comprising one or more substitutions in the VH 75 aggregation prone segment. For example, a variant immunoglobulin may comprise a VH domain with substitutions at positions 61 and 85, and a VL domain with substitutions at position 46. For example, residues 61 in the VH domain may be substituted with K, R, Q, E, D, N and a, preferably R; residue 85 in the VH domain may be substituted with E, D or a, preferably E; residue 46 in the VL domain may be substituted with K, R, H, F or S, preferably R. For example, an immunoglobulin may comprise a VL domain having a L46R substitution, and a VH domain having P61R and V85E substitutions.

A variant immunoglobulin may comprise a VL domain comprising a substitution at the VL 45 aggregation prone segment and a VH domain comprising one or more substitutions at the VH 95 aggregation prone segment and one or more substitutions at the VH CDR3 aggregation prone segment. For example, a variant immunoglobulin may comprise a VH domain with substitutions at positions 95 and 102, and a VL domain with substitutions at positions 37 and 45. For example, residue 95 in the VH domain may be substituted with E or D, preferably D; residue 102 in the VH domain may be substituted with A, F, H, I, L, V or Y, preferably Y; residue 37 in the VL domain may be substituted with Q or N, preferably Q; and residue 45 in the VL domain may be substituted with R, K or E, preferably R. For example, an immunoglobulin may comprise a VL domain with L37Q and Q45R substitutions, and a VH domain with R95D and S102V substitutions.

A variant immunoglobulin may comprise a VL domain comprising a substitution in the VL 45 aggregation prone segment and a VH domain comprising one or more substitutions in the VH 95 aggregation prone segment. For example, a variant immunoglobulin may comprise a VH domain with substitutions at positions 94 and 95, and a VL domain with substitutions at position 46. For example, residue 94 in the VH domain may be substituted with R, K or T, preferably R; residue 95 in the VH domain may be substituted with E or D, preferably D; residue 46 in the VL domain may be substituted with K, R, H, F or S, preferably R. For example, an immunoglobulin may comprise a VL domain having L46R substitutions, and a VH domain having H94R and R95D substitutions.

The techniques required to make substitutions in the amino acid sequences of the aggregation prone segments of the VH, VL and CH1 domains are well known in the art. For example, nucleic acids encoding variant immunoglobulins comprising one or more substitutions may be generated using standard techniques of DNA manipulation and mutagenesis; for example, as Current Protocols in Molecular Biology (modern Molecular Biology techniques), second edition, Ausubel et al. eds., John Wiley & Sons, 1992, or Molecular Cloning: aLaboratory Manual (molecular cloning: A laboratory Manual): second edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. The nucleic acids may then be expressed to produce variant immunoglobulins as described below.

Variant sequences may be tested for their ability to bind to and/or neutralize the target antigen of a parent immunoglobulin and/or for their improved producibility.

The introduction of one or more of the substitutions indicated above may increase the producibility of a variant immunoglobulin relative to its parent immunoglobulin by increasing the producibility upon expression and reducing the aggregation tendency of the variant immunoglobulin relative to its parent immunoglobulin.

For example, a variant immunoglobulin may exhibit at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 200%, or at least 500% increase in productivity relative to a parent immunoglobulin, and at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 90%, or at least 99% decrease in aggregation relative to a parent immunoglobulin. Variant immunoglobulins may exhibit up to 100% reduction in aggregation (i.e., complete elimination of aggregation) relative to the parent immunoglobulin.

Methods of producing the variant immunoglobulins described herein may comprise providing a parent immunoglobulin, introducing a substitution in the VL 37 aggregation-prone segment and/or the VL 45 aggregation-prone segment in the VL domain; and/or

Introducing substitutions in the VH FR1 aggregation prone segment, the VH 95 th aggregation prone segment and the VH CDR3 aggregation prone segment of the VH domain,

thereby producing a variant immunoglobulin, wherein the variant immunoglobulin exhibits increased productivity and reduced aggregation relative to the parent immunoglobulin.

For example, a method of producing a variant immunoglobulin can comprise:

providing a parent immunoglobulin which is capable of producing,

introducing one or more of the following substitutions in the VH domain: a, D, E, Q or V substitution at position 1; r at position 17; r at position 94; a substitution at position 95 with D; a substitution at position 96; a substitution at position 100 with F; f, G, L, M or P substitution at position 100d, preferably F or M substitution; a, F, H, I, L, V or Y substitution at position 102, preferably V or Y substitution; and/or

The following substitutions were introduced in the VL domain: a Q substitution at position 37; e, K, R, Q or V substitution at position 45, preferably K or R substitution; l or Y substitution at position 46, preferably L substitution;

thereby producing a variant immunoglobulin, wherein the variant immunoglobulin exhibits increased productivity and reduced aggregation relative to the parent immunoglobulin.

For example, suitable VH domains may have substitutions at positions 94 and 102 as described above, optionally with other substitutions at positions 94 and 100. For example, residue 95 in the VH domain may be substituted with D, residue 102 may be substituted with A, F, H, I, L, V or Y, preferably V or Y; optionally, residue 94 may also be substituted with R and residue 100 with F.

Suitable VL domains may have substitutions at positions 37 and 45 as described above. For example, the residue at position 37 may be substituted with Q and the residue at position 45 may be substituted with E, K, R, Q or V, preferably K or R. The VL domain may, for example, comprise L37Q and Q45R substitutions.

Variant immunoglobulins may comprise a VH domain with substitutions at positions 94 and 95 and a VL domain with substitutions at position 46 as described above. For example, residues 94 and 95 in the VH domain may be substituted with R and D, respectively, and residue 46 in the VL domain may be substituted with R. For example, an immunoglobulin may comprise a VL domain having L46R substitutions, and a VH domain having H94R and R95D substitutions.

Other variant immunoglobulins may comprise a VH domain with substitutions at positions 95 and 102 and a VL domain with substitutions at positions 37 and 45 as described above. For example, residues 95 and 102 in the VH domain may be substituted with D and V, respectively, and residues 37 and 45 in the VL domain may be substituted with Q and R, respectively. For example, an immunoglobulin may comprise a VL domain with L73Q and Q45R substitutions, and a VH domain with R95D and S102V substitutions.

The presence of aggregation-prone segments described herein can help identify immunoglobulins that exhibit reduced or suboptimal producibility.

The method of evaluating the producibility of an immunoglobulin may comprise:

identifying one or more amino acid residues at a position selected from the group consisting of: 18 th, 20 th, 45 th, 46 th, 74 th, 91 th, 93 th and 96 th positions in the VL domain of the immunoglobulin, 1 st, 5 th, 12 th, 17 th, 19 th, 20 th, 94 th, 96 th, 100c th, 100d th, 102 th and 103 th positions in the VH domain of the immunoglobulin, and 153 th position in the CH1 domain,

wherein in the VL domain of said immunoglobulin, if residue 18 is not R, S or V, preferably not R or V, residue 20 is not K, R, S or V, preferably not R or V, residue 45 is not E, K, R, Q or V, preferably not K or R, residue 46 is not L or Y, preferably not L, residue 74 is not V, residue 91 is not A, F, L, R, S or W, preferably not W, residue 93 is not D, F, L, Q, S, T, W or Y, preferably not Q and/or residue 96 is not E, L, V or W, preferably not V; and/or

In the VH domain of the immunoglobulin, if residue 1 is not A, D, E, Q or V, residue 5 or 12 is not V, residue 17 is not R, residue 19 is not T or V, preferably not T, residue 20 is not A, K, S or T, preferably not a, residue 94 is not R, residue 96 is not a, residue 100c is not D, E, F, G, P, S, T, W or Y, preferably not W, residue 100d is not F, G, L, M or P, preferably not F or M, residue 102 is not A, F, H, I, L, V or Y, preferably not V or Y, residue 103 is not I, L or V; and/or in the CH1 domain of the immunoglobulin if residue 153 is not V; it indicates that the immunoglobulin has suboptimal producibility.

In some embodiments, the amino acid residue at position 100d and/or 102 in a VH domain of an immunoglobulin may be determined. If the residue at position 100d is not F, G, L, M or P, preferably not F or M, and/or the residue at position 102 is not A, F, H, I, L, V or Y, preferably not V or Y, it is an indication that the producibility of the immunoglobulin is suboptimal.

Immunoglobulins identified as having reduced or suboptimal producibility relative to control immunoglobulins can be candidates for modification by the methods described above, thereby producing variant immunoglobulins exhibiting improved producibility.

In particular, one or more residues in the immunoglobulin sequence that adversely affect producibility can be identified. The identified residues can then be altered or modified as described herein, thereby improving producibility.

Another aspect of the invention provides a variant of a parent immunoglobulin produced by the method described above.

Another aspect of the invention provides a variant of a parent immunoglobulin,

wherein the variant immunoglobulin comprises:

a VL domain comprising substitutions relative to the parent immunoglobulin sequence in the framework aggregation prone segment and/or the CDR3 aggregation prone segment;

a VH domain comprising substitutions relative to the parent immunoglobulin sequence in the framework aggregation prone region and/or the CDR3 aggregation prone segment; and/or the presence of a gas in the gas,

a CH1 domain comprising a substitution relative to the parent immunoglobulin sequence in a CH1 aggregation prone region.

Suitable aggregation-prone segments and substitutions are described above.

Substitution at a position in the VL, VH or CH1 domains involves the substitution of the residue at that position in the parent sequence with a different residue, preferably with the residues indicated above. If the parent immunoglobulin already contains the residues indicated above in the amino acid sequence at the appropriate position, this is not a substitution or a substitution residue as described herein. Thus, the parent or germline immunoglobulin is not a variant immunoglobulin as described herein.

As described above, the variant immunoglobulin may have similar or identical biological activity as the parent immunoglobulin, but exhibit improved producibility, e.g., reduced aggregation propensity and/or increased productivity in a heterologous expression system.

For example, a variant immunoglobulin may exhibit an increase in production capacity of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 200%, or at least 500% relative to a parent immunoglobulin, and/or a variant immunoglobulin may exhibit a reduction in aggregation (i.e., a reduction in the proportion of molecules in the ensemble in their native state that undergo aggregation) of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 90%, or at least 99% relative to a parent immunoglobulin. Variant immunoglobulins may exhibit up to 100% reduction in aggregation (i.e., complete elimination of aggregation) relative to the parent immunoglobulin.

As described above, variant immunoglobulins may exhibit improved bioavailability and/or reduced immunogenicity relative to the parent immunoglobulin.

Another aspect of the invention provides an isolated nucleic acid encoding a variant immunoglobulin as described above or a CH1, VH or VL domain thereof.

The nucleic acid may comprise DNA or RNA, and may be fully synthetic or partially synthetic. Unless the context requires otherwise, reference to a nucleotide sequence as described herein includes a DNA molecule having the specified sequence and includes an RNA molecule having the specified sequence, wherein U is replaced by T.

The variant immunoglobulin molecules described herein, or the CH1, VH domain and/or VL domain thereof, may be prepared by the following method: the method comprises expressing the nucleic acid under conditions which result in the production of the variant immunoglobulin molecule or CH1, VH and/or VL domain thereof and recovering the variant immunoglobulin molecule or CH1, VH and/or VL domain thereof.

As described in more detail below, the nucleic acid may encode a variant immunoglobulin or a VH or VL domain of the variant immunoglobulin that lacks one or more CDRs. One or more CDRs obtained from a donor immunoglobulin can be incorporated into the framework of a variant immunoglobulin by inserting suitable nucleic acids encoding the CDRs into a nucleic acid encoding the variant immunoglobulin or a VH or VL domain thereof.

The variant immunoglobulin and the encoding nucleic acid are preferably isolated. Immunoglobulins and nucleic acids are free or substantially free of materials associated therewith, such as other polypeptides or nucleic acids that are present with the immunoglobulins and nucleic acids in the environment in which they are prepared (e.g., cell culture) (if the preparation is by recombinant DNA techniques performed in vitro or in vivo).

Other aspects of the invention provide nucleic acid constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one nucleic acid encoding a variant immunoglobulin described herein or a CH1, VH and/or VL domain thereof.

The constructs may be used in an expression system to express the immunoglobulins described above.

Systems for cloning and expressing immunoglobulins in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines useful in the art for expression of heterologous polypeptides include chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many others. A common preferred bacterial host for small immunoglobulin molecules is e.

Expression of immunoglobulins (e.g., antibodies and antibody fragments) in prokaryotic cells (e.g., E.coli) is well established in the art. For reviews see, for example, Pl ü ckthun, A.Bio/Technology 9: 545-551(1991). Expression in cultured eukaryotic cells is also a useful option for the production of immunoglobulins for those skilled in the art, for a recent review see, e.g., Ref, M.E (1993) curr. opinion biotech.4: 573-; trill j.j.et al (1995) curr.opinion Biotech 6: 553-560. Immunoglobulins, such as antibodies and antibody fragments, may also be expressed in cell-free systems.

Vectors suitable for expressing immunoglobulins can be selected or constructed containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other appropriate sequences. The vector may be a suitable plasmid, virus, e.g. phage or phagemid. For more details, see, for example, Molecular Cloning: aLaboratory Manual (molecular cloning: A Laboratory Manual), second edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. For example, many known techniques and methods for manipulating nucleic acids for nucleic acid construct preparation, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis are described in detail in Current Protocols in Molecular Biology, second edition, Ausubel et al.

The host cell may contain a nucleic acid encoding a variant immunoglobulin or a CH1, VH and/or VL domain thereof. In another aspect, methods are provided that include introducing the nucleic acid into a host cell. The introduction may utilize any available technology. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection and transduction using retroviruses or other viruses (e.g., vaccinia virus), or for insect cells, transduction using baculovirus. Introduction of nucleic acids into host cells, particularly eukaryotic cells, may utilize viral or plasmid based systems. The plasmid system may be maintained as an episome, or may be incorporated into the host cell or into the artificial chromosome. The combination may be performed by random integration or targeted integration of one or more copies into a single or multiple loci. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection with phage.

Following introduction, expression of the nucleic acid may be caused or allowed, for example, by culturing the host cell under conditions suitable for gene expression.

The nucleic acid encoding the variant immunoglobulin or CH1, VH and/or VL domains thereof may be integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by inclusion of sequences that facilitate recombination with the genome, in accordance with standard techniques.

Following production by expression, the variant immunoglobulin or CH1, VH and/or VL domains thereof may be isolated and/or purified using any suitable technique and then used as desired. For example, the method of production may further comprise formulating the product into a composition comprising at least one additional component, such as a pharmaceutically acceptable excipient.

The variant immunoglobulins may be used in methods of treatment or diagnosis of the human or animal body, for example in methods of treatment (which may include prophylactic treatment) of a disease or condition in a human patient, which method comprises administering an effective amount of the variant immunoglobulin to the patient.

Other aspects of the invention provide pharmaceutical compositions comprising a variant immunoglobulin as described herein, methods of preparing a medicament or pharmaceutical composition, and the use of a variant immunoglobulin in the preparation of a medicament for administration, the method comprising formulating the variant immunoglobulin with a pharmaceutically acceptable excipient.

In addition to the variant immunoglobulin, the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other substance known to those skilled in the art. The materials should be non-toxic and should not affect the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration, which may be oral or by injection, for example intravenous injection.

For intravenous injection or injection at the affected site, the active ingredient is in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. One skilled in the art can readily prepare suitable solutions using, for example, isotonic media such as sodium chloride injection, ringer's injection, lactated ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included if desired.

The compositions may be administered alone or in combination with other therapies, which may be simultaneous or sequential, depending on the disease state being treated. Other treatments may include the administration of suitable doses of pain-relieving drugs, such as non-steroidal anti-inflammatory drugs (e.g. aspirin, acetaminophen, ibuprofen or ketoprofen) or opioids (e.g. morphine) or anti-emetics.

Clinical indications for which variant immunoglobulins may be used to provide therapeutic benefit include any disease state or condition for which the parent immunoglobulin may be used.

In accordance with the present invention, the provided compositions can be administered to an individual. Administration is preferably in a "therapeutically effective amount," i.e., an amount sufficient to show benefit to the patient. The benefit may be at least alleviation of at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment (e.g., dosage decisions, etc.) is under the responsibility of general practitioners and other physicians. Suitable dosages of antibodies are well known in the art; see, Ledermann j.a.et al (1991) Int j.cancer 47: 659 and 664; bagshawe k.d.et al (1991) antibodies, Immunoconjugates and Radiopharmaceuticals 4: 915-922.

The exact dosage will depend on a variety of factors, including whether the antibody is for diagnosis or treatment, the size and location of the region to be treated, the exact nature of the antibody (e.g., whole antibody, fragment, or diabody), and the nature of any detectable label or other molecule attached to the antibody. In general, typical antibody doses are: it is administered systemically in an amount of 0.5mg-1g, and topically in an amount of 10 μ g-1 mg. Typically, the antibody is a whole antibody, preferably of the IgG4 isotype. The above are doses for a single treatment of an adult patient, which can be scaled for children and infants, and other antibody formats can also be scaled for molecular weight. The physician may repeat the treatment at daily, twice-weekly, or monthly intervals, as judged.

The variant immunoglobulins and encoding nucleic acids described herein comprising substitutions in aggregation-prone segments may be used in antibody engineering methods to produce immunoglobulins with improved producibility.

Recombinant DNA technology can be used to produce immunoglobulins with variants having improved producibility. The techniques can involve introducing DNA encoding one or more CDR sequences of a donor antibody into a variable region of a variant immunoglobulin described herein. See, for example, EP-A-184187, GB 2188638A or EP-A-239400 and ｃA number of subsequent documents. Alternatively, immunoglobulin-producing hybridomas or other cells can be subjected to genetic mutations in the sequences encoding immunoglobulin aggregation-prone segments, which can improve the producibility of the produced immunoglobulins. This can be used to engineer or reconstitute antibodies with producibility suitable for recombinant production.

In some embodiments, the VH and VL domains of the variant immunoglobulin may lack one or more CDR sequences (e.g., CDR 3). Heterologous CDRs or synthetic CDRs can be introduced into VH and/or VL domains of variant immunoglobulins that lack the corresponding CDR (e.g., CDR3) using recombinant DNA techniques. This can alter the binding properties of the variant immunoglobulin without affecting producibility.

For example, one or more CDR sequences from a donor immunoglobulin (e.g., CDR3) can be used to replace a corresponding CDR sequence in a variant immunoglobulin described herein. 1, 2, 3, 4, 5, or all 6 CDR sequences from the human variant immunoglobulins described herein may be substituted with CDR sequences obtained from a non-human donor immunoglobulin (e.g., a murine immunoglobulin) to produce a humanized immunoglobulin. Humanized immunoglobulins may have the same binding activity as non-human donor immunoglobulins but exhibit reduced immunogenicity in humans and thus may have therapeutic efficacy.

Methods of making hybrid immunoglobulins that bind to a target antigen can include:

providing a donor immunoglobulin capable of binding to said target antigen,

providing the variant immunoglobulin described above, and

replacing 1, 2, 3, 4, 5 or 6 CDR sequences of the variant immunoglobulin with corresponding CDR sequences from the donor immunoglobulin,

thereby producing hybrid immunoglobulins that bind to the target antigen.

Preferably, the VH CDR3 and/or VL CDR3 of the variant immunoglobulin is replaced with the corresponding VH CDR3 and/or VL CDR3 from the donor immunoglobulin.

The variant immunoglobulin may be a human immunoglobulin, and the donor immunoglobulin may be a non-human immunoglobulin.

Techniques suitable for humanizing immunoglobulins by CDR grafting are well known in the art (see, e.g., Riechman et al (1988) Nature 332323-327; Queen et al PNAS USA (1989) 8610029-10033).

The VL CDR3 of the hybrid immunoglobulin may comprise a substitution in the VL CDR 3N-terminal aggregation-prone segment or the VL CDR 3C-terminal aggregation-prone segment described above.

The VH CDR3 of the hybrid immunoglobulin may comprise substitutions in the VH CDR3 aggregation prone segments described above.

Suitable variant immunoglobulins may comprise one or more substitutions in the framework or constant regions described above.

In some embodiments, substitutions may be introduced in the donor VH and/or VL CDR3 sequences. Substitutions may be made before or after insertion of the variant immunoglobulin using standard techniques.

The variant immunoglobulins described herein may also be used in humanization methods other than CDR grafting. One or more of the substitutions described herein may be introduced into the immunoglobulin during the production of the humanized antibody, thereby improving its producibility. Useful humanization techniques are well known in the art (see, e.g., Padlan et al Mol Immunol (1991) 28489-498).

The variant immunoglobulins described herein may be used to generate libraries or libraries for isolating binding specificities.

The method of preparing a variant immunoglobulin library may comprise:

providing a population of variant immunoglobulins as described above,

replacing 1, 2, 3, 4, 5 or 6 CDR sequences of the population of variant immunoglobulins with a library of CDR sequences,

thereby generating a library of variant immunoglobulins.

The variant immunoglobulin library will contain a library of different replacement CDR sequences in the same variant immunoglobulin background.

The repertoire can be screened for variant immunoglobulins that are specific for the target antigen.

In some preferred embodiments, the VH CDR3 of the variant immunoglobulin population may be substituted with a VH CDR3 sequence repertoire. For example, a method of preparing a variant immunoglobulin that binds a target antigen can comprise:

(a) providing a starting population of nucleic acids encoding a VH domain comprising CDR3 to be replaced or lacking a CDR3 encoding region; wherein the VH domain comprises one or more substitutions in one or more of the aggregation-prone segments described above,

(b) combining the VH domain population with a population of donor nucleic acids encoding a VH CDR3 amino acid sequence repertoire such that the donor nucleic acids are inserted into the CDR3 regions of the VH domain population, thereby providing a product population of nucleic acids encoding a VH domain repertoire comprising the VH CDR3 sequences;

(c) nucleic acids expressing the product population;

(d) selecting an immunoglobulin specific for a target antigen; and

(e) recovering the immunoglobulin or nucleic acid encoding the same.

The population of nucleic acids encoding the VH domain may further comprise a nucleotide sequence encoding a CH1 domain, the CH1 domain comprising one or more substitutions in the CH1 aggregation prone segment described above.

In the same manner, a library of VL CDR3 sequences may be combined with a population of nucleic acids encoding VL domains that comprise one or more substitutions in the aggregation prone segments described herein and comprise the VL CDR3 to be replaced or lack the VL CDR3 encoding region.

In addition to CDR3, libraries of CDR1 and CDR2 sequences can also be grafted into populations of VH and/or VL domains comprising one or more substitutions in the aggregation-prone segments described herein, and then screened for variant immunoglobulins specific for the target antigen.

The CDR-derived sequence repertoire can be shuffled with a population of VH or VL domains described herein that lack the corresponding CDR sequences, and the shuffled complete VH or VL domains combined with a homologous VL or VH domain (which may also comprise one or more substitutions as described herein), to provide an engineered variant immunoglobulin. The library can then be displayed in a suitable host system (e.g.the phage display system of WO 92/01047) so that suitable immunoglobulins with improved producibility can be selected. The library may be composed of a library from 10⁴More than one individual member (e.g. 10)⁶To 10⁸Or 10¹⁰Individual members).

Techniques for CDR shuffling and antibody engineering are well known in the art and those skilled in the art are able to use these techniques using conventional methods to provide immunoglobulins with improved producibility.

The CDR sequence library may comprise a plurality of residues at one or more positions in the CDR sequence. For example, a CDR sequence library can include a plurality of residues at 1, 2, 3, 4, 5, or more positions in a CDR sequence.

The library of CDR sequences can include one or more variable positions in the CDR sequences. For example, a CDR sequence library can include 1, 2, 3, 4, 5, or more variable positions in the CDR sequence. The residue at each variable position may be different between different members of the library. The residues at the variable positions may be random (e.g., they may be any naturally occurring amino acid with the same probability) or may be selected from a predetermined set or subset of amino acids. For example, the VLCDR3 of a variant immunoglobulin can be substituted with a repertoire of donor VL CDR3 sequences that comprise A, F, L, R, S and W at position 91, D, F, L, Q, S, T, W and Y at position 93, and/or E, L, V and W at position 96. The VH CDR3 of the variant immunoglobulin may be substituted with a repertoire of donor VH CDR3 sequences comprising D, E, F, G, P, S, T, W and Y at position 100c, F, G, L, P and M at position 100d, A, F, H, I, L, V and Y at position 102, and/or I, L and V at position 103.

Biasing the CDR3 repertoire toward these residues at these positions can help improve producibility.

In addition to one or more variable positions, the library of CDR sequences may also include one or more non-variable positions. The residue at each non-variable position may be the same in each member of the library.

The incorporation of a library of CDR sequences into a receptor variant immunoglobulin can be used to generate a library of variant immunoglobulins with different binding properties, each member of the library comprising one or more substitutions in one or more aggregation-prone segments described herein.

The library of variant immunoglobulins may comprise:

a VL domain comprising a substitution in the framework aggregation prone segment and/or CDR3 aggregation prone region described above;

a VH domain comprising a substitution in the framework aggregation prone region and/or CDR3 aggregation prone segment described above; and/or the presence of a gas in the gas,

a CH1 domain comprising a substitution in the CH1 aggregation prone region described above;

wherein the members of the library differ in one or more CDR sequences.

Libraries of variant immunoglobulins can be used to identify immunoglobulins with improved producibility that bind to a target antigen. Methods of obtaining one or more immunoglobulins that bind to a target antigen can include:

contacting the immunoglobulin repertoire described above with the antigen, and

selecting one or more immunoglobulins in the pool that are capable of binding the antigen.

The repertoire can, for example, be displayed on the surface of phage particles, each particle containing nucleic acids encoding an antibody VH variable domain displayed on its surface, and optionally a displayed VL domain (if present).

After selecting an immunoglobulin capable of binding an antigen and being displayed on a phage particle, nucleic acids may be obtained from the phage particle displaying said immunoglobulin. The nucleic acids having the nucleic acid sequences obtained from the phage particles displaying the immunoglobulin can be used for the subsequent production of immunoglobulins or antibody VH variable domains and/or VL domains thereof by expression.

Immunoglobulins may be tested for their ability to bind to an antigen, their ability to compete with other immunoglobulins for binding to an antigen, and/or their ability to neutralize an antigen. Under appropriate conditions, the binding affinity and neutralizing capacity of different immunoglobulins can be compared. The producibility of immunoglobulins can also be tested.

Various other aspects and embodiments of the invention will be apparent to those skilled in the art in view of this disclosure. All documents mentioned in this specification are herein incorporated in their entirety by reference.

As used herein, "and/or" should be understood to mean that the specific disclosure of each of the two specified features or components, may or may not have the other. For example, "a and/or B" should be understood as a specific disclosure of each of (i) a, (ii) B, and (iii) a and B, as if each were individually listed herein.

Unless the context dictates otherwise, the description and definitions of the features shown above are not limited to any particular aspect or embodiment of the invention and may apply equally to all aspects and embodiments described.

Certain aspects and embodiments of the invention will now be described by way of example in conjunction with the tables set forth below.

Tables 1a and 1b show the correspondence between Kabat and honeyger numbering schemes for immunoglobulin VH and VL domains.

Tables 2a and 2b show examples of amino acid substitutions in aggregation prone segments that improve producibility. Preferred substitutions are also indicated. The standard IUPAC-IUB JCBN amino acid one letter abbreviations are used.

Tables 3a and 3b show examples of combinations of substitutions in aggregation-prone segments that improve producibility.

Table 4 shows the producibility of examples of variant immunoglobulins.

Experiment of

Gene synthesis

Sequences were optimized and synthesized by commercial suppliers and subcloned into pee6.4 and pee 12.4. DNA stocks were prepared by transformation into Top10 cells, O/N culture of the cells in LB medium, and purification using the QIAGEN Endofree plasmid purification kit. The DNA was resuspended in TE buffer and the concentration was determined using a Nanodrop spectrophotometer.

Conventional cell culture

CHOK1SV cells were subcultured every 3-4 days in CD-CHO medium supplemented with 15mM L-glutamine and in CO₂In a shaker (140rpm), at 36.5 ℃ and 10% CO₂Incubate at 85% humidity.

Transient transfection

CHOK1SV cells were transfected with Lipofectamine 2000. 72 hours after transfection, the supernatant was collected, centrifuged and stored at 4 ℃ before analysis.

Stable transfection

Large scale expression was performed under MSX selection using stably transfected CHOK1SV cells. After collection, the supernatant was stored at 4 ℃ before purification.

Purification of

The supernatant was protein a purified using HiTrap column (GE) and stored at 4 ℃ prior to concentration and buffer exchange.

Antibody concentration and buffer exchange

The sample was concentrated by centrifugation at 2000g for 15-20 minutes. The material was buffer exchanged 4-5 times with formulation buffer (50mM phosphate, 100mM NaCl, pH 7.4). After buffer exchange, the samples were diluted in formulation buffer to the appropriate working concentration.

GP-HPLC

Replicate samples were analyzed by GP-HPLC on an Agilent 1200 series HPLC system. An 80. mu.l aliquot of the 1mg/ml sample (stored at 4 ℃) was loaded and run at 1 ml/min for 15 minutes. The results were analyzed using chemstation software.

IgG titre

Antibody expression yield was determined by sandwich ELISA. Titers were normalized to light chain specific controls.

Thioflavin T binding

Thioflavin T can be used to monitor aggregation. 1mg/ml of purified antibody was incubated at 60 ℃ for 1 hour, then mixed with thioflavin T at a final concentration of 30. mu.M, and fluorescence was measured at Ex 305 and Em 508nm (Hawe, A., Sutter, M and Jiskoot, W.pharmaceutical Research 200825(7) 1487-99).

ANS bonding

8-anilino-1-naphthalenesulfonic Acid (ANS) can be used to monitor aggregation. 1mg/ml of purified antibody was incubated at 60 ℃ for 1 hour, then mixed with ANS at a final concentration of 30. mu.M, and fluorescence was measured at Ex-360, Em/508nm (Hawe, A., Sutter, M and Jiskoot, W.PharmaceuticalResearch 200825(7) 1487-99).

Results

Immunoglobulin sequences were analyzed by in silico and in vitro methods to identify aggregation-prone segments. The identified aggregation-prone segments are shown in Table 2

Substitutions in the aggregation prone segments identified in the VH and VL domains (as shown in SEQ ID NOS: 1 and 2) that reduce aggregation propensity were designed and synthesized (Table 2). The effect of substitutions on expression, stability and aggregation of immunoglobulins was tested (tables 3 and 4).

It has been found that substitution of residues in the identified aggregation-prone segments enables improved producibility, in particular enables reduced aggregation-prone and/or improved levels of production.

Other numbered statements of the invention:

1. a variant immunoglobulin comprising:

a light chain Variable (VL) domain comprising substitutions in a Framework (FR) aggregation-prone segment and/or a complementarity determining region 3(CDR3) aggregation-prone segment;

a heavy chain Variable (VH) domain comprising substitutions in the framework region aggregation prone segment and/or the CDR3 aggregation prone segment; and/or

A heavy chain constant region 1(CH1) domain comprising a substitution in a CH1 aggregation prone segment.

2. A method of improving producibility of an immunoglobulin comprising:

the identification of the parent immunoglobulin is carried out,

introducing a substitution in the framework aggregation-prone segment and/or the CDR3 aggregation-prone segment in the VL domain of the parent immunoglobulin, and/or

Introducing substitutions in framework region aggregation-prone segments and/or CDR3 aggregation-prone segments in the parent immunoglobulin VH domain, and/or

Introducing a substitution in a constant region aggregation-prone segment in the CH1 domain of the parent immunoglobulin,

thereby producing a variant immunoglobulin having improved producibility relative to the parent immunoglobulin.

3. The variant immunoglobulin or method of any preceding claim, wherein the variant immunoglobulin has a reduced propensity for aggregation and/or increased production capacity when expressed relative to the parent immunoglobulin.

4. The variant immunoglobulin or method of any preceding claim, wherein the framework aggregation-prone segment of the VL domain is selected from the group consisting of: a 20 th aggregation propensity segment, a 45 th aggregation propensity segment, and a 74 th aggregation propensity segment.

5. The variant immunoglobulin or method as claimed in statement 4, wherein said aggregation propensity segment at position 20 extends from position 15 to position 23 of said VL domain.

6. The variant immunoglobulin or method as set forth in statement 5, wherein the VL domain comprises a substitution at position 18 thereof.

7. The variant immunoglobulin or method as claimed in statement 6, wherein the residue at position 18 is substituted with R, S or V.

8. The variant immunoglobulin or method as claimed in statement 7, wherein the VL domain comprises T18R, T18S, or T18V.

9. The variant immunoglobulin or the method as claimed in any one of claims 5-8, wherein said VL domain comprises a substitution at position 20 thereof.

10. The variant immunoglobulin or method as claimed in statement 9, wherein the residue at position 20 is substituted with K, R, S or V.

11. The variant immunoglobulin or method as claimed in statement 10, wherein the VL comprises a T20K, T20R, T20S, or T20V substitution.

12. The variant immunoglobulin or the method as claimed in any of claims 4-11, wherein said aggregation propensity segment at position 45 extends from position 42 to position 49 of said VL domain.

13. The variant immunoglobulin or method as claimed in claim 12, wherein said VL domain comprises a substitution at position 45.

14. The variant immunoglobulin or method as claimed in statement 13, wherein the residue at position 45 is substituted with E, K, R, Q or V.

15. The variant immunoglobulin or method as claimed in statement 14, wherein the VL domain comprises a T45E, T45K, T45R, T45Q, or T45V substitution.

16. The variant immunoglobulin or the method as claimed in any one of claims 12-15, wherein said VL domain comprises a substitution at position 46.

17. The variant immunoglobulin or method as claimed in statement 16, wherein the residue at position 46 is substituted with L or Y.

18. The variant immunoglobulin or method as claimed in claim 17, wherein the VL domain comprises a T46L or T46Y substitution.

19. A variant immunoglobulin or a method as claimed in any of claims 4-18, wherein said aggregation propensity segment at position 74 extends from position 71 to position 77 of said VL domain sequence.

20. The variant immunoglobulin or method as claimed in claim 19, wherein said VL domain comprises a substitution at position 74.

21. The variant immunoglobulin or method as claimed in claim 20, wherein the residue at position 74 is substituted with V.

22. A variant immunoglobulin or method as described in statement 21, wherein the VL domain comprises a T74V substitution.

23. The variant immunoglobulin or method of any preceding claim, wherein the VL domain CDR3 aggregation-prone segment is selected from the group consisting of: a VL CDR 3N-terminal aggregation-prone segment and a VL CDR 3C-terminal aggregation-prone segment.

24. The variant immunoglobulin or method as claimed in statement 23, wherein the VL CDR3N terminal aggregation prone segment extends from residue C88 to amino acid 8 at the end of residue C88C in the VL domain sequence.

25. The variant immunoglobulin or method as claimed in statement 24, wherein the VL domain comprises a substitution at one or more of positions 88, 89, 90, 91, 92, 93, 94, 95 or 95 a.

26. The variant immunoglobulin or method as claimed in claim 25, wherein said VL domain comprises a substitution at position 91.

27. The variant immunoglobulin or method as claimed in statement 26, wherein the residue at position 91 is substituted with A, F, L, R, S or W.

28. The variant immunoglobulin or the method as claimed in statement 27, wherein the VL domain comprises a Y91A, Y91F, Y91L, Y91R, Y91S, or Y91W substitution.

29. The variant immunoglobulin or method as claimed in any of claims 24-29, wherein said VL domain comprises a substitution at position 93.

30. The variant immunoglobulin or method as claimed in statement 29, wherein the residue at position 93 is substituted with D, F, L, Q, S, T, W or Y.

31. The variant immunoglobulin or method as claimed in statement 30, wherein the VL domain comprises a P93D, P93F, P93L, P93Q, P93S, P93T, P93W, or P93Y substitution.

32. A variant immunoglobulin or a method as claimed in claim 23, wherein said VL C-terminal CDR3 aggregation prone segment extends from amino acid 3 to amino acid 99 of the N-terminus of residue F98 in the VL domain sequence.

33. The variant immunoglobulin or method as claimed in claim 32, wherein said VL domain comprises a substitution at one or more of position 95, 96, 97, 98 or 99.

34. The variant immunoglobulin or method as claimed in claim 33, wherein said VL domain comprises a substitution at position 96.

35. The variant immunoglobulin or method as claimed in statement 34, wherein the residue at position 96 is substituted with E, L, V or W.

36. The variant immunoglobulin or the method as claimed in statement 35, wherein the VL domain comprises a R96E, R96L, R96V, or R96W substitution.

37. The variant immunoglobulin or method of any preceding claim, wherein the VH domain framework aggregation-prone segment is selected from: FR1 aggregation prone segment, 95 th aggregation prone segment and 102 th aggregation prone segment.

38. A variant immunoglobulin or method as claimed in claim 37, wherein said FR1 aggregation propensity segment is located in framework region 1 of said VH domain sequence.

39. A variant immunoglobulin or method as claimed in statement 38, wherein said FR1 aggregation-prone segment is an aggregation-prone segment selected from the group consisting of: 1 st to 3 rd bits, 4 th to 6 th bits, 11 th to 13 th bits, and 16 th to 21 th bits.

40. A variant immunoglobulin or method as described in statement 39, wherein the FR1 aggregation propensity segment extends from position 1 to position 3.

41. The variant immunoglobulin or method as claimed in statement 40, wherein said VH domain comprises a substitution at position 1 thereof.

42. The variant immunoglobulin or method as described in statement 41, wherein the residue at position 1 is substituted with E, Q, D, A or V.

43. The variant immunoglobulin or method as claimed in statement 42, wherein the VH domain comprises a Q1E, Q1D, Q1A, or Q1V substitution or an E1Q, E1D, E1A, or E1V substitution.

44. A variant immunoglobulin or method as claimed in statement 39, wherein said FR1 aggregation propensity segment extends from position 4 to position 6 of said VH domain.

45. The variant immunoglobulin or method as claimed in statement 44, wherein said VH domain comprises a substitution at its 5 th position.

46. The variant immunoglobulin or method as described in statement 45, wherein the residue at position 5 is substituted with V.

47. The variant immunoglobulin or method as claimed in statement 46, wherein said VH domain comprises L5V.

48. A variant immunoglobulin or method as claimed in statement 39, wherein said FR1 aggregation propensity segment extends from position 11 to position 13 of said VH domain.

49. The variant immunoglobulin or method as claimed in statement 48, wherein said VH domain comprises a substitution at position 12 thereof.

50. The variant immunoglobulin or method as claimed in statement 49, wherein the residue at position 12 is substituted with V.

51. A variant immunoglobulin or method as claimed in claim 50, wherein the VH domain comprises L12V.

52. A variant immunoglobulin or method as claimed in statement 39, wherein said FR1 aggregation propensity segment extends from position 16 to position 21 of said VH domain.

53. The variant immunoglobulin or method as claimed in statement 52, wherein said VH domain comprises a substitution at position 17.

54. The variant immunoglobulin or method as claimed in statement 53, wherein the residue at position 17 is substituted with R.

55. The variant immunoglobulin or method as claimed in statement 54, wherein said VH domain comprises a G17R substitution.

56. The variant immunoglobulin or method of any of claims 52-55, wherein the VH domain comprises a substitution at position 19.

57. The variant immunoglobulin or method as described in statement 56, wherein the residue at position 19 is substituted with a T or a V.

58. The variant immunoglobulin or the method as claimed in statement 57, wherein said VH domain comprises L19T or and L19V substitutions.

59. The variant immunoglobulin or method of any of claims 52-58, wherein said VH domain comprises a substitution at position 20.

60. The variant immunoglobulin or method as claimed in statement 59, wherein the residue at position 20 is substituted with A, K, S or T.

61. The variant immunoglobulin or method as claimed in statement 60, wherein the position of the residue wherein the VH domain comprises a R20A, R20K or R20S or R20T substitution.

62. The variant immunoglobulin or method of any of claims 37-61, wherein said aggregation-prone segment at position 95 extends from position 91 to position 99.

63. The variant immunoglobulin or method as claimed in statement 62, wherein the VH domain comprises a substitution at one or more of position 91, 92, 93, 94, 95, 96, 97, 98 and/or 99.

64. The variant immunoglobulin or method as claimed in statement 63, wherein the VH domain comprises a substitution at position 94.

65. The variant immunoglobulin or method as claimed in statement 64, wherein the residue at position 94 is substituted with R.

66. A variant immunoglobulin or method as described in statement 65, wherein said VH domain comprises a K94R substitution.

67. The variant immunoglobulin or method of any of claims 63-66, wherein the VH domain comprises a substitution at position 96.

68. The variant immunoglobulin or method as claimed in claim 67, wherein the residue at position 96 is substituted with A.

69. A variant immunoglobulin or method as described in statement 68, wherein said VH domain comprises a G96A substitution.

70. The variant immunoglobulin or method of any of claims 1-69, wherein said VH CDR3 aggregation prone segment extends from position 100c to position 103.

71. The variant immunoglobulin or method as claimed in statement 70, wherein the VH domain comprises a substitution at one or more of positions 100c, 100d, 101, 102 and 103.

72. The variant immunoglobulin or method as set forth in statement 71, wherein said VH domain comprises a substitution at position 100 c.

73. The variant immunoglobulin or method as described in statement 72, wherein the residue at position 100c is substituted with D, E, F, G, P, S, T, W or Y.

74. The variant immunoglobulin or the method as claimed in statement 73, wherein said VH domain comprises a100cD, a100cE, a100cF, a100cG, a100cP, a100cS, a100cT, or a100cW or a100 cY.

75. The variant immunoglobulin or method of any of claims 70-74, wherein the VH domain comprises a substitution at position 100 d.

76. The variant immunoglobulin or method as claimed in statement 75, wherein the residue at position 100d is substituted with F, G, L, P or M.

77. The variant immunoglobulin or the method as claimed in statement 76, wherein the VH domain comprises a S100dF, S100dG, S100dL, S100dP, or S100dM substitution.

78. The variant immunoglobulin or method of any of claims 70-77, wherein the VH domain comprises a substitution at position 102.

79. The variant immunoglobulin or method as claimed in statement 78, wherein the residue at position 102 is substituted with A, F, H, I, L, V or Y.

80. The variant immunoglobulin or method as described in statement 79, wherein the VH domain comprises a P102A, P102F, P102H, P102I, P102L, P102Y, or P102V substitution.

81. The variant immunoglobulin or method of any of claims 70-80, wherein said VH domain comprises a substitution at position 103.

82. The variant immunoglobulin or method as claimed in statement 81, wherein the residue at position 103 is substituted with I, L or V.

83. The variant immunoglobulin or method as claimed in statement 82, wherein the VH domain comprises a W103I, W103L, or W103V substitution.

84. The variant immunoglobulin or method of any one of statements 1-83, wherein the CH1 aggregation propensity segment extends from position 150 to position 156.

85. The variant immunoglobulin or the method as described in statement 84, wherein the CH1 domain comprises a substitution at position 153.

86. The variant immunoglobulin or the method as described in statement 85, wherein the CH1 domain comprises a substitution at position 153.

87. The variant immunoglobulin or method as claimed in statement 86, wherein the residue at position 153 is substituted with V.

88. A variant immunoglobulin or method as described in statement 87, wherein the VH domain comprises a S153V substitution.

89. The variant immunoglobulin or method of any of the preceding claims, wherein the sequence of the variant immunoglobulin has up to 10 amino acid substitutions relative to the parent immunoglobulin sequence.

90. The variant immunoglobulin or method of any preceding claim, wherein the sequence of the variant immunoglobulin comprises a substitution at 2 in the VL FR1 and/or FR2 domains; comprises 3 substitutions in the VLCDR3 domain; comprises 2 substitutions in the VL FR2 domain and 3 substitutions in the VL CDR3 domain; comprises 2 substitutions in the VL FR1 domain, 2 substitutions in the VL FR2 domain and 3 substitutions in the VL CDR3 domain; comprises a substitution at 2 in the VH FR1 domain; comprises 1 substitution in the VH FR1 domain and 1 substitution in the VH CDR3 domain; comprises 2 substitutions in the VH CDR3 domain; or comprises 1 substitution in the CH1 domain and 1 substitution in the VH FR1 domain.

91. A variant immunoglobulin or method as described in statement 90, wherein the sequence of said VH domain comprises substitutions at 100D (D101-1) and 102.

92. The variant immunoglobulin or the method as claimed in claim 91, wherein the residue at position 102 is substituted with A, F, H, I, L, V or Y, and wherein the residue at position 100d is substituted with F, G, L, P or M.

93. The variant immunoglobulin or method as claimed in statement 92, wherein the VH domain comprises a P102A, P102F, P102H, P102I, P102L, P102Y, or P102V substitution and a S100dF, S100dG, S100dL, S100dP, or S100dM substitution.

94. The variant immunoglobulin or method as claimed in statement 93, wherein the VH domain comprises S100dF and P102V substitutions, S100dM and P102V substitutions, S100dF and P102Y substitutions, or S100dM and P102Y substitutions.

95. The variant immunoglobulin or method of any of the preceding claims, wherein the variant immunoglobulin is humanized.

96. A method of evaluating producibility of an immunoglobulin, comprising:

identifying one or more amino acid residues at a position selected from the group consisting of: 18 th, 20 th, 45 th, 46 th, 74 th, 91 th, 93 th and 96 th positions in the VL domain of the immunoglobulin, 1 st, 5 th, 12 th, 17 th, 19 th, 20 th, 94 th, 96 th, 100c th, 100d th, 102 th and 103 th positions in the VH domain of the immunoglobulin, and 153 th position in the CH1 domain of the immunoglobulin,

wherein in the VL domain of said immunoglobulin, if residue 18 is not R, S or V, preferably not R or V, residue 20 is not K, R, S or V, preferably not R or V, residue 45 is not E, K, R, Q or V, preferably not K or R, residue 46 is not L or Y, preferably not L, residue 74 is not V, residue 91 is not A, F, L, R, S or W, preferably not W, residue 93 is not D, F, L, Q, S, T, W or Y, preferably not Q and/or residue 96 is not E, L, V or W, preferably not V; and/or

In the VH domain of the immunoglobulin, if residue 1 is not A, D, E, Q or V, residue 5 or 12 is not V, residue 17 is not R, residue 19 is not T or V, preferably not T, residue 20 is not A, K, S or T, preferably not a, residue 94 is not R, residue 96 is not a, residue 100c is not D, E, F, G, P, S, T, W or Y, preferably not W, residue 100d is not F, G, L, M or P, preferably not F or M, residue 102 is not A, F, H, I, L, V or Y, preferably not V or Y, residue 103 is not I, L or V; and/or

In the CH1 domain of the immunoglobulin, if residue 153 is not V;

it indicates that the producibility of the immunoglobulin is suboptimal.

97. A method of evaluating producibility of an immunoglobulin, comprising:

identifying amino acid residues at position 100d and/or 102 in the VH domain of the immunoglobulin,

wherein if position 100d is S and/or position 102 is P, it is indicative that the immunoglobulin has suboptimal producibility.

98. An isolated nucleic acid encoding the variant immunoglobulin of any one of claims 1-95, or encoding a VH domain, a VL domain, or a CH1 domain comprising one or more of the substitutions.

99. An expression vector comprising the isolated nucleic acid of statement 98.

100. A host cell comprising the expression vector of statement 99.

101. A method of making a variant immunoglobulin comprising:

expressing the nucleic acid of statement 98 under conditions that result in production of the variant immunoglobulin or VH domain, VL domain or CH1 domain thereof, and recovering the variant immunoglobulin or VH domain, VL domain or CH1 domain thereof.

102. The method of claim 101, comprising formulating the variant immunoglobulin in a composition comprising a pharmaceutically acceptable excipient.

103. A pharmaceutical composition comprising the variant immunoglobulin of any of claims 1-95 and a pharmaceutically acceptable excipient.

104. A variant immunoglobulin according to any one of claims 1-95 for use as a medicament.

105. A method of making a hybrid immunoglobulin capable of binding a target antigen, comprising:

providing a donor immunoglobulin capable of binding to said target antigen,

providing a variant immunoglobulin of any one of claims 1-89,

replacing one or more CDR sequences of the variant immunoglobulin with corresponding CDR sequences of the donor immunoglobulin,

thereby producing hybrid immunoglobulins that bind to the target antigen.

106. The method of statement 105, wherein the VL CDR3 of the hybrid immunoglobulin comprises a substitution in the VL CDR 3N-terminal aggregation-prone segment or the VL CDR 3C-terminal aggregation-prone segment.

107. The method of claim 105 or 106, wherein the VHCDR3 of the hybrid immunoglobulin comprises a substitution in a VH CDR3 aggregation-prone segment.

108. A method of preparing a library of variant immunoglobulins comprising:

providing a variant immunoglobulin of any of claims 1-95,

replacing one or more CDR sequences of the variant immunoglobulin with a CDR sequence comprising a random residue at one or more positions therein,

thereby generating a library with multiple variant immunoglobulins.

109. The method of statement 108, wherein VL CDR3 of the variant immunoglobulin is substituted with a random CDR3 sequence, the random CDR3 sequence comprising A, F, L, R, S or W at position 91, D, F, L, Q, S, T, W or Y at position 93, and/or E, L, V or W at position 96.

110. The method of statement 108 or 109, wherein the VHCDR3 of the variant immunoglobulin is substituted with a random CDR3 sequence, which random CDR3 sequence comprises D, E, F, G, P, S, T, W or Y at position 100c, F, G, L, P or M at position 100d, A, F, H, I, L, V or Y at position 102, and/or I, L or V at position 103.

111. A library of variant immunoglobulins wherein each member of the library comprises:

a VL domain comprising a substitution in the framework aggregation-prone segment and/or the CDR3 aggregation-prone segment;

a VH domain comprising a substitution in the framework region aggregation prone segment and/or the CDR3 aggregation prone segment; and/or

A CH1 domain comprising a substitution in a CH1 aggregation prone segment,

wherein the members of the library are different in one or more CDR sequences.

112. A population of nucleic acids encoding the library of variant immunoglobulins according to statement 111.

113. A method for obtaining one or more immunoglobulins capable of binding a target antigen with improved producibility comprising:

contacting the library of variant immunoglobulins of statement 111 with the antigen, and

selecting one or more immunoglobulins capable of binding said antigen in said repertoire.

The sequence is as follows:

VL

NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDPEMRVFGGGTKLTVL(SEQ ID NO：1)

VH

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTYEGPTYFASDPWGQGTLVTVSS(SEQ ID NO：2)

table 1A: VL Domain numbering

Sequence B	Kabat	Honegger	Sequence B	Kabat	Honegger
						N	1	1	V	33	41
F	2	2	Q	34	42
						M	3	3	W	35	43
L	4	4	Y	36	44
						T	5	5	Q	37	45
Q	6	6	Q	38	46
						P	7	7	R	39	47
	8	8	P	40	48
						H	9	9	G	41	49
S	10	10	S	42	50
						V	11	11	S	43	51
S	12	12	P	44	52
						E	13	13	T	45	53
S	14	14	T	46	54
						P	15	15	V	47	55
G	16	16	I	48	56
						K	17	17	Y	49	57
T	18	18	E	50	58
						V	19	19			59
T	20	20			60
						I	21	21			61
S	22	22			62
						C	23	23			63

T	24	24			64
						R	25	25			65
S	26	26			66
						S	27	27	D	51	67
G	27a	28	N	52	68
						S	27b	29	Q	53	69
	27c	30	R	54	70
							27d	31	P	55	71
	27e	32	S	56	72
							27f	33	G	57	73
		34	V	58	74
								35	P	59	75
I	28	36	D	60	76
						A	29	37	R	61	77
S	30	38	F	62	78
						N	31	39	S	63	79
Y	32	40	G	64	80
						Sequence B	Kabat	Honegger	Sequence B	Kabat	Honegger
S	65	81			121
						I	66	82			122
D	67	83			123
						S	68	84			124
S	68a	85			125
						S	68b	86			126
N	69	87			127
						S	70	88			128
A	71	89			129
						S	72	90			130
L	73	91			131
						T	74	92			132
I	75	93			133
						S	76	94			134
G	77	95			135
						L	78	96			136
K	79	97	R	96	137
						T	80	98	V	97	138
E	81	99	F	98	139
						D	82	100	G	99	140
E	83	101	G	100	141
						A	84	102	G	101	142
D	85	103	T	102	143
						Y	86	104	K	103	144
Y	87	105	L	104	145
						C	88	106	T	105	146
Q	89	107	V	106	147
						S	90	108	L	107	148
Y	91	109
						D	92	110
P	93	111
						E	94	112
M	95	113
							95a	114
	95b	115
							95c	116
	95d	117
							95e	118
	95f	119
								120

Table 1B: VH Domain numbering

Sequence B	Kabat	Honegger	Sequence B	Kabat	Honegger
						Q	1	1			41
V	2	2			42
						Q	3	3	W	36	43
L	4	4	V	37	44
						V	5	5	R	38	45
E	6	6	Q	39	46
						S	7	7	A	40	47
	8	8	P	41	48
						G	9	9	G	42	49
G	10	10	K	43	50
						G	11	11	G	44	51
V	12	12	L	45	52
						V	13	13	E	46	53
Q	14	14	W	47	54
						P	15	15	V	48	55
G	16	16	A	49	56
						R	17	17	V	50	57
S	18	18	I	51	58
						L	19	19	S	52	59
R	20	20	Y	52a	60
						L	21	21		52b	61
S	22	22		52c	62
						C	23	23			63
A	24	24	D	53	64
						A	25	25	G	54	65
S	26	26	S	55	66
						G	27	27	N	56	67
F	28	28	K	57	68
						T	29	29	Y	58	69
F	30	30	Y	59	70
						S	31	31	A	60	71
S	32	32	D	61	72
						Y	33	33	S	62	73
G	34	34	V	63	74
						M	35	35	K	64	75
H	35a	36	G	65	76
							35b	37	R	66	77
		38	F	67	78
								39	T	68	79
		40	I	69	80
						Sequence B	Kabat	Honegger	Sequence B	Kabat	Honegger
S	70	81		100g	121
						R	71	82		100h	122
D	72	83		100i	123
						N	73	84			124
S	74	85			125
						K	75	86			126
N	76	87			127
						T	77	88			128
L	78	89			129
						Y	79	90			130
L	80	91			131
						Q	81	92			132
M	82	93			133
						N	82a	94			134
S	82b	95			135
						L	82c	96			136
R	83	97	D	101	137
						A	84	98	P	102	138
E	85	99	W	103	139
						D	86	100	G	104	140
T	87	101	Q	105	141
						A	88	102	G	106	142
V	89	103	T	107	143
						Y	90	104	L	108	144
Y	91	105	V	109	145
						C	92	106	T	110	146
A	93	107	V	111	147
						K	94	108	S	112	148
T	95	109	S	113	149
						Y	96	110
E	97	111
						G	98	112
P	99	113
						T	100	114
Y	100a	115
						F	100b	116
A	100c	117
						S	100d	118
	100e	119
							100f	120

TABLE 2a

^*VL CDR3 position: y91, P93 are also numbered as distance from Kabat C88;

r96 is also numbered as a distance from Kabat's F98.

^**VH CDR3 position: due to the heterogeneity of the CDRs 3 (heavy chains), the positions also agreed

The distance of D101 (unchanged in Kabat) is named.

TABLE 2b

TABLE 3a

^*VL CDR3 position: y91, P93 are also numbered as distance from Kabat C88;

r96 is also numbered as a distance from Kabat's F98.

^**VH CDR3 position: due to the heterogeneity of the CDRs 3 (heavy chains), the positions also agreed

The distance of D101 (unchanged in Kabat) is named.

TABLE 3b

Variant combinations	Combinations of tests
		HC
R95 S102	R95D S102V
		H94R95 V100 S102	H94R R95D V100F S102V
HC/LC
		P61 V85/L46	P61R V85E/L46R
R95 S102/L37 Q45	R95D S102V/L37Q Q45R
		H94 R95/L46	H94R R95D/L46R
LC
		L37 Q45	L37Q Q45R

TABLE 4

VL CDR3 position: y91, P93 are also numbered as the distance from Kabat C88, and R96 is also numbered as the distance from Kabat F98.

^**VH CDR3 position: due to the heterogeneity of the CDRs 3 (heavy chains), positions were also named by distance from D101 (unchanged in Kabat).

All data shown are relative to wild type.

Claims (58)

1. A method of improving producibility of an immunoglobulin comprising:

identifying a parent immunoglobulin having a VL domain and a VH domain,

introducing a substitution in the framework aggregation-prone segment and/or the CDR3 aggregation-prone segment in the VL domain of the parent immunoglobulin, and/or

Introducing substitutions in framework region aggregation-prone segments and/or CDR3 aggregation-prone segments in the VH domain of the parent immunoglobulin, and/or

Introducing a substitution in a constant region aggregation-prone segment in the CH1 domain of the parent immunoglobulin,

thereby producing a variant immunoglobulin having a VL domain and a VH domain that exhibits improved producibility relative to the parent immunoglobulin.

2. A variant immunoglobulin comprising:

a light chain Variable (VL) domain comprising a substitution in a Framework (FR) aggregation-prone segment and/or a complementarity determining region 3(CDR3) aggregation-prone segment;

a heavy chain Variable (VH) domain comprising a substitution in the framework region aggregation prone segment and/or the CDR3 aggregation prone segment; and/or the presence of a gas in the gas,

a heavy chain constant region 1(CH1) domain comprising a substitution in a CH1 aggregation prone segment.

3. The method of claim 1 or the variant immunoglobulin of claim 2, wherein the variant immunoglobulin exhibits a reduced propensity for aggregation and increased productivity upon expression relative to the parent immunoglobulin.

4. The variant immunoglobulin or method of claim 3, wherein the variant immunoglobulin exhibits at least a 20% increase in production capacity and at least a 30% decrease in aggregation propensity relative to the parent immunoglobulin.

5. The variant immunoglobulin or method of claim 3 or 4, wherein a substitution is introduced in the VL 37 aggregation-prone segment and/or the VL 45 aggregation-prone segment in the VL domain of the parent immunoglobulin; and/or

Substitutions are introduced in the VH FR1 aggregation prone segment, the VH 95 th aggregation prone segment and the VH CDR3 aggregation prone segment in the VH domain of the parent immunoglobulin.

6. The variant immunoglobulin or method of claim 5, wherein the substitution is introduced at a position selected from position 1, 17, 94, 95, 96, 100d and 102 of the VH domain sequence and/or at a position selected from position 37, 45 and 46 of the VL domain sequence.

7. The variant immunoglobulin or method of claim 6, wherein the residue at position 37 in the VL domain sequence is substituted with Q or N.

8. The variant immunoglobulin or method of claim 7, wherein the VL domain comprises an L37Q substitution.

9. The variant immunoglobulin or method of any of claims 6-8, wherein residue 45 of the VL domain sequence is substituted with E, K, R, Q or V.

10. The variant immunoglobulin or method of claim 9, wherein the VL domain comprises a T45E, T45K, T45R, T45Q, or T45V substitution.

11. The variant immunoglobulin or method of any of claims 6-10, wherein residue 46 of the VL domain sequence is substituted with L or Y.

12. The variant immunoglobulin or method of claim 11, wherein the VL domain comprises a T46L or T46Y substitution.

13. The variant immunoglobulin or method of any of claims 6-12, wherein the residue at position 1 in the VH domain sequence is substituted with E, Q, D, A or V.

14. The variant immunoglobulin or method of claim 13, wherein the VH domain comprises a Q1E, Q1D, Q1A, or Q1V substitution, or an E1Q, E1D, E1A, or E1V substitution.

15. The variant immunoglobulin or method of any of claims 6-14, wherein the residue at position 17 in the VH domain sequence is substituted with R.

16. The variant immunoglobulin or method of claim 15, wherein the VH domain comprises a G17R substitution.

17. The variant immunoglobulin or method of any of claims 6-16, wherein residue 94 of the VH domain sequence is substituted with R.

18. The variant immunoglobulin or method of claim 17, wherein the VH domain comprises an H94R substitution.

19. The variant immunoglobulin or method of any of claims 6-18, wherein residue 95 in the VH domain sequence is substituted with D.

20. The variant immunoglobulin or method of claim 19, wherein the VH domain comprises a R95D substitution.

21. The variant immunoglobulin or method of any of claims 6-20, wherein the residue at position 96 in the VH domain sequence is substituted with a.

22. The variant immunoglobulin or method of claim 21, wherein the VH domain comprises a G96A substitution.

23. The variant immunoglobulin or method of any of claims 6-22, wherein the residue at position 100d in the VH domain sequence is substituted with F, G, L, P or M.

24. The variant immunoglobulin or method of claim 23, wherein the VH domain comprises a S100dF, S100dG, S100dL, S100dP, or S100dM substitution.

25. The variant immunoglobulin or method of any of claims 6-24, wherein the residue at position 102 in the VH domain sequence is substituted with A, F, H, I, L, V or Y.

26. The variant immunoglobulin or method of claim 25, wherein the VH domain comprises a P102A, P102F, P102H, P102I, P102L, P102Y, P102V, or S102V substitution.

27. The variant immunoglobulin or method of any of claims 3-26, wherein the sequence of the variant immunoglobulin has up to 10 amino acid substitutions relative to the parent immunoglobulin sequence.

28. The variant immunoglobulin or method of any of claims 3-27, wherein the sequence of the VH domain comprises substitutions in a VH FR1 aggregation prone segment and a VH CDR3 aggregation prone segment.

29. The variant immunoglobulin or method of claim 28, wherein the sequence of the VH domain comprises substitutions at positions 1 and 102.

30. The variant immunoglobulin or method of claim 29, wherein the residue at position 102 is substituted with A, F, H, I, L, V or Y, and wherein the residue at position 1 is substituted with E, Q, D, A or V.

31. The variant immunoglobulin or method of claim 30, wherein the VH domain comprises a P102Y substitution and a Q1A or Q1E substitution.

32. The variant immunoglobulin or method of any of claims 3-31, wherein the sequence of the VH domain comprises a substitution in the VH 95 aggregation prone segment and the VH CDR3 aggregation prone segment.

33. The variant immunoglobulin or method of claim 32, wherein the sequence of the VH domain comprises substitutions at positions 95 and 102.

34. The variant immunoglobulin or method of claim 33, wherein the residue at position 102 is substituted with A, F, H, I, L, V or Y, and wherein the residue at position 95 is substituted with E or D.

35. The variant immunoglobulin or method of claim 34, wherein the VH domain comprises a S102V substitution and a R95D substitution.

36. The variant immunoglobulin or method of any of claims 33-35, wherein the VH domain further comprises substitutions at positions 94 and 100.

37. The variant immunoglobulin or method of claim 36, wherein the residue at position 94 is substituted with R and the residue at position 100 is substituted with F.

38. The variant immunoglobulin or method of claim 37, wherein the VH domain comprises a H94R substitution and a V100F substitution.

39. The variant immunoglobulin or method of any of claims 3-38, wherein the VL domain sequence of the variant immunoglobulin comprises a substitution at aggregation-prone segments 37 and 45.

40. The variant immunoglobulin or method of claim 39, wherein the VL domain sequence of the variant immunoglobulin comprises substitutions at positions 37 and 45.

41. The variant immunoglobulin or method of claim 40, wherein the residue at position 37 is substituted with Q or N, and wherein the residue at position 45 is substituted with R, K or E.

42. The variant immunoglobulin or method of claim 41, wherein the VL domain comprises an L37Q substitution and a Q45R substitution.

43. The variant immunoglobulin or method of any of claims 3-42, wherein the VL domain comprises an S102V substitution and an R95D substitution, and the VL domain comprises an L37Q substitution and a Q45R substitution.

44. The variant immunoglobulin or method of any of claims 3-43, wherein the VH domain comprises one or more substitutions in the VH aggregation prone segment at position 95 and the VL domain comprises one or more substitutions in the aggregation prone segment at position 45.

45. The variant immunoglobulin or method of claim 44, wherein the VH domain comprises a substitution at positions 94 and 95 and the VL domain comprises a substitution at position 46.

46. The variant immunoglobulin or method of claim 45, wherein the residue at position 94 in the VH domain is substituted with R, K or T, and the residue at position 95 in the VH domain is substituted with E or D, and the residue at position 46 in the VL domain is substituted with K, R, H, F or S.

47. The variant immunoglobulin or method of claim 46, wherein the VH domain comprises H94R and R95D substitutions, and the VL domain comprises an L46R substitution.

48. The variant immunoglobulin or method of any of claims 3-47, wherein the VH domain of the variant immunoglobulin comprises two or more substitutions in the VH CDR3 aggregation-prone segment.

49. The variant immunoglobulin or method of claim 48, wherein the VH domain comprises a substitution at positions 100d and 102.

50. The variant immunoglobulin or method of claim 49, wherein the VH domain comprises S100dF and P102V substitutions; s100dM and P102V substitutions; s100dF and P102Y substitutions; or S100dM and P102Y substitutions.

51. The variant immunoglobulin or method of any of the preceding claims, wherein the variant immunoglobulin is humanized.

52. The variant immunoglobulin or method of any of the preceding claims, wherein the variant immunoglobulin is expressed in a recombinant system.

53. The variant immunoglobulin or method of any of the preceding claims, wherein the variant immunoglobulin is isolated and/or purified after expression.

54. The variant immunoglobulin or method of any of the preceding claims, wherein the variant immunoglobulin is formulated with a pharmaceutically acceptable excipient.

55. A method of evaluating producibility of an immunoglobulin, comprising:

identifying one or more amino acid residues at a position selected from the group consisting of: 37 th, 45 th and 46 th positions in a VL domain, 1 st, 17 th, 94 th, 95 th, 96 th, 100d th and 102 th positions in a VH domain of the immunoglobulin,

wherein in the VL domain of said immunoglobulin, if residue 37 is not Q, residue 45 is not E, K, R, Q or V, preferably not K or R, residue 46 is not L or Y, preferably not L; and/or

In the VH domain of the immunoglobulin, if residue 1 is not A, D, E, Q or V, residue 17 is not R, residue 94 is not R, residue 96 is not a, residue 100d is not F, G, L, M or P, preferably not F or M, residue 102 is not A, F, H, I, L, V or Y, preferably not V or Y;

it indicates that the producibility of the immunoglobulin is suboptimal.

56. The method of claim 55 comprising said immunoglobulin producibility being suboptimal and said immunoglobulin producibility being improved by the method of any one of claims 1 and 3 to 54.

57. A method of making an immunoglobulin with improved producibility comprising:

providing a nucleic acid encoding a variant immunoglobulin produced by the method of any one of claims 1 and 3 to 46,

expressing said nucleic acid under conditions which result in the production of said variant immunoglobulin, and recovering said variant immunoglobulin.

58. The method of claim 57, comprising formulating the variant immunoglobulin in a composition comprising a pharmaceutically acceptable excipient.