Method for generating a single variable domain of immunoglobulin

Abstract

To provide methods for the production of polypeptides comprising at least one immunoglobulin variable domain that result in an increased yield.SOLUTION: A method for the production of a polypeptide comprising or essentially consisting of at least one immunoglobulin single variable domain is provided, the method comprising the step of expressing, in a Pichia host, the polypeptide and simultaneously enhancing, in the Pichia host, the expression of the auxiliary protein HAC1 spliced type.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a method for producing immunoglobulin monovariable domains. More specifically, the present invention provides an improved method for producing immunoglobulin monovariable domains that yield increased yields. The present invention further provides nucleic acids, gene constructs and host cells for use in the method of the present invention, as well as immunoglobulin monovariable domains that can be obtained by the method of the present invention.

[0002] Background technology For therapeutic application, the quality of antibody or antibody fragment products must be extremely high. This places high demands on the production process of biological therapeutic agents. The production costs of these therapeutic compounds are strongly influenced by the difficulties encountered during the production process. Low yields or lack of uniformity will affect the economy of the production process and, therefore, the overall cost of the therapeutic agent.

[0003] The limits for obtaining adequate yields of functional products have been reported for conventional immunoglobulins and their fragments in a wide range of expression systems, including baculovirus systems in in vitro translation, Escherichia coli (E. coli), yeast (e.g., Saccharomyces cerevisiae and Pichia pastoris), mammalian cells (e.g., Chinese hamster ovary cells), and insect cells. In particular, the impairments in antibody expression appear to be due to insufficient supply of light chains, improper processing and folding in the endoplasmic reticulum (ER), and intracellular accumulation of heavy chain fragments (Lange et al. 2001, J. Immunol. Methods 255: 103; Gasser et al. 2006, Biotechnol Bioeng. 94: 353; Gach et al. 2007, J. Biotechnol. 128: 735; Jenkins et al. 2009, Biotechnol. Appl. Biochem. 53: 73).

[0004] Intervention in protein folding and secretion pathways has been described as one of various strategies for improving the expression and quality of recombinant proteins such as monoclonal antibodies (MAbs). However, overexpression of one or more components of the ER secretion mechanism has yielded mixed results regarding productivity improvements. Many challenges remain in achieving consistently high yields in biopharmaceutical production (Jenkins et al. 2009, Biotechnol. Appl. Biochem. 53: 73).

[0005] Pichia pastris was developed as a host for the synthesis of heterologous proteins. While this host is known as a highly efficient expression system, it has been found to exhibit a considerably low success rate, particularly in the synthesis of complex proteins. In P. pastris, co-overexpression of immunoglobulin-binding protein (BiP) increased the secretion level of scFv (A33scFv) by approximately threefold. In contrast, co-overexpression of protein disulfide isomerase (PDI1) had no apparent effect on A33scFv secretion. Co-overexpression of BiP and PDI1 in P. pastris did not increase A33scFv secretion, and protein levels remained the same as in the control strain (Damasceno et al. 2007, Appl. Microbiol. Biotechnol. 74: 381). Compared to the control strain, the productivity of the 2F5 Fab fragment in P. pastris was improved from 1.2 times with co-overexpression with BFR2 to 2.3 times with overexpression of SSE1 or KIN2 (Gasser et al. 2007, Appl. Environ. Microbiol. 73: 6499). Overexpression of the basic leucine zipper (bZIP) transcription factor HAC1 had only a slight effect on the secretion of the Mab 2F5 Fab fragment in P. pastris (1.3 times), while overexpression of PDI1 enabled a 1.9-fold increase in Fab levels (Gasser et al. 2006, Biotechnol Bioeng. 94: 353). The authors conclude that sufficient light chain supply and interchain disulfide bond formation may be the main rate-limiting factors for Fab association and subsequent secretion.

[0006] In contrast to the difficulties observed with conventional quadruple-chain antibodies or their fragments (including Fab and scFv), immunoglobulin monovariable domains are known to be readily expressed and secreted from hosts such as E. coli and P. pastris at sufficient rates and levels. Immunoglobulin monovariable domains lack interchain disulfide crosslinks and are characterized by the formation of antigen-binding sites by the monoimmunoglobulin monovariable domain, which do not require interaction with further domains (e.g., in the form of VH / VL interactions) for antigen recognition. For example, the generation of nanobodies as one specific example of immunoglobulin monovariable domains in prokaryotic hosts such as E. coli has been described in numerous studies (see, e.g., Ghahroudi et al. 1997, FEBS Letters 414: 521-526; Muyldermans 2001, J. Biotechnol. 74: 277-302; Vranken et al. 2002, Biochemistry 41: 8570-8579).

[0007] The generation of nanobodies in lower eukaryotic hosts such as P. pastris is described in Frenken et al. 2000 (J. Biotechnol. 78: 11-21), International Publication No. 94 / 25591, International Publication No. 2010 / 125187, International Publication No. 2012 / 056000, and International Publication No. 2012 / 152823.

[0008] Recombinant camel single variable domains are routinely obtained at levels of 5–10 mg / l when expressed in E. coli grown in shaking culture flasks without any optimization of conditions (Ghahroudi et al. 1997). Higher yields of VHH expression are even possible using other expression systems. Production levels of 9.3 mg / l / OD660 or approximately 250 mg of secreted protein per liter of Saccharomyces yeast culture in a shaking flask were described by Frenken et al. 2000. More recently, yields of nanobody exceeding 1 g per liter have been reported when expressed in P. pastris (International Publication No. 2010 / 139808, International Publication No. 2012 / 152823).

[0009] International Publication No. 2010 / 125187 describes a method for generating a single variable domain in yeast (e.g., P. pastris). The method in International Publication No. 2010 / 125187 applies conditions that promote the formation of disulfide crosslinks in the single variable domain. One of the proposed conditions is the enhancement of thiol isomerase (e.g., PDI1) expression.

[0010] The fact that fully functional immunoglobulin single variable domains can be easily generated at sufficient speed and level in, for example, E. coli or yeast, demonstrates a significant advantage of this immunoglobulin format over conventional immunoglobulins.

[0011] Summary of the Invention VHH and nanobodies can be expressed using expression systems described in the art (International Publication No. 1994 / 25591; Ghahroudi et al. 1997, FEBS Letters 414: 521-526; Frenken et al. 2000, J. Biotechnol. 78: 11-21; Muyldermans 2001, J. Biotechnol. 74: 277-302; Vranken et al. 2002, Biochemistry 41: 8570-8579; International Publication No. 2010 / 125187; International Publication No. 2012 / 056000; International Publication No. 2012 / 152823 and Ablynx (Other patent applications by NV) The inventors have found that, in some cases (e.g., polyvalent VHHs and nanobodies, and / or VHHs and nanobodies having one or more disulfide crosslinks), the expression of VHHs and nanobodies is more difficult, resulting in much lower expression levels and / or yields than expected. For example, the inventors unexpectedly observed problems with the generation of several therapeutic VHH1 type immunoglobulin monovariable domains. When these immunoglobulin monovariable domains were expressed in P. pastris, the inventors obtained these immunoglobulin monovariable domains in much lower yields compared to the yields typically obtained with VHH2 and VHH3 type immunoglobulin monovariable domains. In contrast to what is established with immunoglobulin monovariable domains, the expression of certain immunoglobulin monovariable domains in P. pastris, such as VHH1 type immunoglobulin monovariable domains, did not yield large amounts of functional products.

[0012] The inventors have provided solutions to this problem, as further described herein. Furthermore, the inventors have observed that the proposed solutions for improving the yield of immunoglobulin monovariable domains are generally applicable, that is, applicable not only to improving the yield of VHH1 type immunoglobulin monovariable domains, but also to improving other immunoglobulin monovariable domains, such as VHH2 and VHH3 type immunoglobulin monovariable domains.

[0013] Therefore, in one aspect, the present invention relates to the observation of low yields when a specific immunoglobulin monovariate domain is expressed in P. pastris.

[0014] In a further aspect of the present invention, a method for generating immunoglobulin monovariable domains is provided that increases the yield of the resulting product. These methods are also referred to herein as “Methods(s) of the Present Invention.” Thus, the present invention also provides a method for generating immunoglobulin monovariable domains that overcomes this unexpected problem. More specifically, the inventors (by screening a library of auxiliary proteins) discovered that enhancing the expression of certain auxiliary proteins (PDI1 (SEQ ID NO: 5), Kar2p (SEQ ID NO: 4), RPP0 (SEQ ID NO: 6), and especially the HAC1 splicing type (SEQ ID NO: 14)) in a Pichia host increased the yield of immunoglobulin monovariable domains when expressed in the Pichia host. Yield increases of more than 2 to more than 10 (and even more) were obtained.

[0015] In one embodiment, therefore, the present invention relates to a method for increasing the expression and / or production yield of a single immunoglobulin variable domain in Pichia (e.g., P. pastris). In the method of the present invention, a single immunoglobulin variable domain is expressed, but at the same time, the expression of the HAC1 splicing type is also enhanced.

[0016] The immunoglobulin monovariate domains used in the method of the present invention may form part of a polypeptide (also referred to as the "polypeptide of the present invention") which may comprise or substantially comprise one or more (i.e., at least one) immunoglobulin monovariate domains and may further comprise one or more additional amino acid sequences (all of which may be linked via one or more suitable linkers).

[0017] Accordingly, the present invention provides a method for generating a polypeptide (hereinafter referred to as the “Polypeptide of the Invention”) in a pichia host (e.g., P. pastris) comprising at least one immunoglobulin monovariate domain or substantially thereof, the method comprising the steps of expressing the polypeptide of the Invention in a pichia host and enhancing the expression of a HAC1 splicing protein in the pichia host. The method of the Invention may further comprise the steps of isolating and / or purifying the polypeptide of the Invention.

[0018] The method of the present invention is particularly suitable for immunoglobulin monovariable domains and polypeptides of the present invention that are not readily expressible in Pichia (e.g., P. pastris) or are expressed in very low yields when expressed under standard conditions (as further defined herein), and therefore, when these immunoglobulin monovariable domains or polypeptides of the present invention are expressed in Pichia (e.g., P. pastris), it is not possible to obtain a sufficient amount of immunoglobulin monovariable domains and / or polypeptides. Accordingly, in one embodiment, the immunoglobulin monovariable domains and / or polypeptides of the present invention are selected from immunoglobulin monovariable domains and / or polypeptides that, when expressed in a Pichia host (e.g., P. pastris) under standard Pichia expression conditions (as further defined herein), yields of 0.5 g / l or less, for example, 0.4 g / L, 0.3 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, 0.01 g / L, or even less. In another embodiment, the immunoglobulin monovariable domains and / or polypeptides of the present invention are selected from immunoglobulin monovariable domains and / or polypeptides such that the yield obtained when expressed in a pichia host (e.g., P. pastris) under pichia expression conditions (as further defined herein) is inversely correlated (as further defined herein) with the copy number of the nucleic acid encoding the immunoglobulin monovariable domain and / or polypeptide.

[0019] In specific embodiments of the present invention, the method of the present invention is particularly suitable for the immunoglobulin monovariable domains and polypeptides of the present invention that can be readily expressed in Pichia such as P. pastris. Accordingly, in one embodiment, the immunoglobulin monovariable domains and / or polypeptides of the present invention are selected from immunoglobulin monovariable domains and / or polypeptides that, when expressed in a Pichia host (e.g., P. pastris) under Pichia expression conditions (as further defined herein), yield 0.5 g / l or more, for example, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, or even higher.

[0020] In the method described above, the polypeptide of the present invention may contain or substantially consist of two or more immunoglobulin monovariable domains. Such polypeptides are also referred to as polyvalent polypeptides. Therefore, in specific embodiments, the polypeptide of the present invention to be expressed by the method of the present invention is a polyvalent polypeptide.

[0021] Alternatively, the polypeptide expressed by the method of the present invention may contain or substantially consist of a single immunoglobulin monovariate domain. Such a polypeptide will also be referred to herein as a monovalent polypeptide. Thus, in another specific embodiment, the polypeptide of the present invention to be expressed by the method of the present invention is a monovalent polypeptide.

[0022] In the above method, the immunoglobulin single variable domain (which may be present in the polypeptide of the present invention) is an immunoglobulin single variable domain that is a light chain variable domain or a heavy chain variable domain, more specifically a heavy chain variable domain derived from a conventional four-chain antibody or a heavy chain variable domain derived from a heavy chain antibody, particularly a domain antibody (or an amino acid sequence suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), "dAb" (or an amino acid sequence suitable for use as dAb), or a nanobody (including but not limited to a VHH sequence), and preferably can be a nanobody (but not limited thereto).

[0023] In a preferred embodiment, the nanobody expressed in the method of the present invention is a nanobody obtained by techniques such as a VHH sequence (VHH), a (partially) humanized VHH sequence (humanized VHH), a camelized heavy chain variable domain (camelized VH), or affinity maturation.

[0024] In another specific embodiment of the present invention, the immunoglobulin single variable domain (which may be present in the polypeptide of the present invention) expressed in the method of the present invention contains at least two disulfide bridges. In another specific embodiment, the immunoglobulin single variable domain belongs to the group of VHH1-type immunoglobulin single variable domains.

[0025] In another embodiment, the immunoglobulin single variable domain (which may be present in the polypeptide of the present invention) does not belong to the group of VHH1-type immunoglobulin single variable domains, but belongs to another group of immunoglobulin single variable domains, for example, VHH2, VHH3, or any other type of immunoglobulin single variable domain.

[0026] In yet another specific embodiment, the immunoglobulin single variable domain expressed in the method of the present invention (which may be present in the polypeptide of the present invention), for example, the immunoglobulin single variable domain described in International Publication No. WO 2012 / 042026 and International Publication No. WO 2013 / 045707 specifically binds to c-Met. Therefore, the polypeptide of the present invention comprising at least one immunoglobulin single variable domain that specifically binds to c-Met (where the immunoglobulin single variable domain herein comprises two disulfide bridges) forms a specific but non-limiting embodiment of the present invention. A specific nanobody for use in the method of the present invention is SEQ ID NO: 49.

[0027] In a preferred embodiment, one or more immunoglobulin single variable domain(s) (which may be present in the polypeptide of the present invention) expressed in the method of the present invention, for example, the immunoglobulin single variable domain described in US Provisional Application No. 62 / 254,375 of Ablynx NV (see also PCT / EP2016 / 077595), specifically binds to TNF.

[0028] Therefore, the polypeptide of the present invention comprising one or more (at least one) immunoglobulin single variable domains that specifically bind to TNF forms a specific and non-limiting embodiment of the present invention.

[0029] In a specific embodiment, the polypeptide of the present invention expressed in the method of the present invention comprises or consists essentially of one immunoglobulin single variable domain. Such a polypeptide will be referred to herein as a monovalent polypeptide. Therefore, in a preferred embodiment, in the method of the present invention, the polypeptide is a monovalent polypeptide.

[0030] Such a monovalent polypeptide comprising or consisting essentially of one immunoglobulin single variable domain may comprise at least two disulfide bridges. Therefore, the immunoglobulin single variable domain that specifically binds to TNF belongs to the VHH1 type immunoglobulin single variable domain.

[0031] Alternatively, the immunoglobulin monovariable domain contains one disulfide crosslink. Therefore, in a preferred embodiment, an immunoglobulin monovariable domain that specifically binds to TNF does not belong to the group of VHH1 type immunoglobulin monovariable domains, but belongs to another group of immunoglobulin monovariable domains, such as VHH2, VHH3, or any other type of immunoglobulin monovariable domain.

[0032] In a preferred embodiment, in the method of the present invention, the immunoglobulin monovariable domain substantially consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), where CDR1 is sequence number 58, CDR2 is sequence number 60, and CDR3 is sequence number 62.

[0033] Specific nanobodies for use in the method of the present invention are selected from the group consisting of SEQ ID NOs. 55 and 56.

[0034] The polypeptides of the present invention, comprising or substantially comprising one or more immunoglobulin monovariable domains, may further comprise one or more other residues or binding units, optionally linked via one or more peptide linkers.

[0035] In one embodiment, one or more of the aforementioned other residues may be effective in preventing or reducing the binding of antibodies already present in serum (so-called “existing antibodies”) to the polypeptide of the present invention (as further described herein).

[0036] In another embodiment, the one or more other residues or binding units may also be selected from the group consisting of an immunoglobulin monovariable domain, a domain antibody, an amino acid sequence suitable for use as a domain antibody, a single domain antibody, an amino acid sequence suitable for use as a single domain antibody, a "dAb", an amino acid sequence suitable for use as a dAb, or a nanobody. A polypeptide comprising or substantially comprising two or more binding units is also referred to as a polyvalent construct.

[0037] In specific embodiments of the present invention, the polypeptide comprising or substantially comprising one or more immunoglobulin monovariable domains expressed in the method of the present invention is a polyvalent construct.

[0038] In another specific embodiment, the polypeptide of the present invention is a divalent, trivalent, or tetravalent polypeptide.

[0039] In certain embodiments of the present invention, the one or more other binding units described above may provide a polypeptide of the present invention having an extended half-life compared to a polypeptide that does not contain the one or more binding units described above. The one or more other binding units that provide a polypeptide with an extended half-life may be selected from the group consisting of binding units that can bind to serum albumin (e.g., human serum albumin) or serum immunoglobulin (e.g., IgG), but are not limited to the following.

[0040] In the method of the present invention, the expression of HAC1 splicing type can be enhanced by introducing one or more nucleic acids encoding the HAC1 splicing type protein into a pichia host. In another embodiment, the expression of HAC1 splicing type protein can be enhanced by introducing one or more potent promoters controlling the expression of nucleic acids encoding the HAC1 splicing type protein into a pichia host.

[0041] A polypeptide comprising or substantially comprising at least one immunoglobulin monovariate domain and a HAC1 splicing protein may be expressed from the same gene construct. In this particular aspect of the present invention, the transcription of the nucleic acid encoding the polypeptide comprising or substantially comprising at least one immunoglobulin monovariate domain and the transcription of the nucleic acid encoding the HAC1 splicing protein may be controlled by the same promoter or by different promoters. The nucleic acid encoding the HAC1 splicing protein may be located on a gene construct downstream of the nucleic acid encoding the polypeptide comprising or substantially comprising at least one immunoglobulin monovariate domain. Alternatively, the nucleic acid encoding the polypeptide comprising or substantially comprising at least one immunoglobulin monovariate domain may be located on a gene construct downstream of the nucleic acid encoding the HAC1 splicing protein.

[0042] In another aspect of the present invention, the polypeptide comprising or substantially comprising at least one immunoglobulin monovariate domain and the HAC1 splicing protein may be expressed from different gene constructs. In this particular aspect of the present invention, the transcription of nucleic acids encoding the polypeptide comprising or substantially comprising at least one immunoglobulin monovariate domain and the transcription of nucleic acids encoding the HAC1 splicing protein from different gene constructs may be controlled by two separate promoters, which may be the same or different.

[0043] Polypeptides and / or HAC1 splicing proteins comprising or substantially comprising at least one immunoglobulin monovariate domain may also be expressed from chromosomes. In this particular embodiment, the expression of HAC1 splicing proteins may be enhanced by introducing a strong promoter into the chromosomes of the Pichia host. Alternatively, one or more nucleic acids encoding HAC1 splicing proteins controlled by a strong promoter may be introduced into the chromosomes of the Pichia host. Or, one or more nucleic acids encoding polypeptides comprising or substantially comprising at least one immunoglobulin monovariate domain may be introduced into the chromosomes of the Pichia host.

[0044] In one aspect of the present invention, the number of nucleic acids (groups) encoding polypeptides containing or substantially comprising a single immunoglobulin variable domain is 1. In another aspect of the present invention, the number of nucleic acids (groups) encoding polypeptides containing or substantially comprising a single immunoglobulin variable domain is 2 or more.

[0045] In another aspect, the present invention also relates to nucleic acids encoding the polypeptide (or a suitable fragment thereof) of the present invention and encoding a HAC1 splicing protein. Such nucleic acids are also referred to herein as “the nucleic acids of the present invention” and may be in the form of, for example, gene constructs as further described herein.

[0046] Accordingly, the present invention also relates to nucleic acids of the present invention in the form of a gene construct. Such a gene construct is also referred to herein as the “gene construct of the present invention,” and therefore comprises nucleic acids encoding the polypeptide of the present invention and nucleic acids encoding the HAC1 splicing protein. In a specific embodiment, in such a gene construct of the present invention, the nucleic acid encoding the HAC1 splicing protein is located downstream of the nucleic acid encoding the polypeptide of the present invention. In another specific embodiment, in such a gene construct of the present invention, the nucleic acid encoding the polypeptide of the present invention is located downstream of the nucleic acid encoding the HAC1 splicing protein. In the gene construct of the present invention, the expression of the nucleic acid encoding the polypeptide of the present invention and the expression of the nucleic acid encoding the HAC1 splicing protein may be controlled by the same promoter or by different promoters. The promoter may be a constitutive promoter or an inductive promoter.

[0047] In one aspect of the present invention, the copy number of the nucleic acid encoding the polypeptide of the present invention is 1. In another aspect of the present invention, the copy number of the nucleic acid encoding the polypeptide of the present invention is 2 or more.

[0048] In one aspect of the present invention, the number of auxiliary proteins is one, and the auxiliary protein is of the HAC1 splicing type. In another aspect of the present invention, the expression of one or more auxiliary proteins is enhanced. In yet another aspect of the present invention, the additional auxiliary proteins(s) are selected from PDI1, Kar2p, and RPP0. In yet another aspect of the present invention, the number of auxiliary proteins is two. In yet another aspect of the present invention, the number of auxiliary proteins is greater than two, for example, three or more. In yet another aspect, the two or more auxiliary proteins are selected from the following combinations of auxiliary proteins: -PDI1 and HAC1 splicing type; -Kar2p and HAC1 splicing types; -RPP0 and HAC1 splicing type; -PDI1, Kar2p, and HAC1 splicing types; -PDI1, RPP0, and HAC1 splicing types; -Kar2p, RPP0 and HAC1 splicing types; and -PDI1, Kar2p, RPP0, and HAC1 splicing types.

[0049] In a preferred embodiment, the expression of the following accessory proteins is enhanced: -PDI1, Kar2p, and HAC1 splicing type; or -Kar2p, RPP0, and HAC1 splicing type.

[0050] In a preferred embodiment of the present invention, the auxiliary protein is a HAC1 splicing type (SEQ ID NO: 14).

[0051] In another preferred embodiment, the additional accessory protein is selected from PDI1 (SEQ ID NO: 5), Kar2p (SEQ ID NO: 4), RPP0 (SEQ ID NO: 6), and HAC1 splicing type (SEQ ID NO: 14).

[0052] In another preferred embodiment, two or more accessory proteins are selected from the following combinations of accessory proteins: -PDI1 (sequence number 5) and Hac1 splicing type (sequence number 14); -Kar2p (sequence number 4) and Hac1 splicing type (sequence number 14); -RPP0 (sequence number 6) and Hac1 splicing type (sequence number 14); -PDI1 (sequence number 5), Kar2p (sequence number 4), and Hac1 splicing type (sequence number 14); -PDI1 (sequence number 5), RPP0 (sequence number 6), and Hac1 splicing type (sequence number 14); -Kar2p (sequence number 4), RPP0 (sequence number 6), and Hac1 splicing type (sequence number 14); and -PDI1 (SEQ ID NO: 5), Kar2p (SEQ ID NO: 4), RPP0 (SEQ ID NO: 6), and Hac1 splicing type (SEQ ID NO: 14).

[0053] In another aspect, the present invention relates to the introduction of the nucleic acids and gene constructs of the present invention into a pichia host, also referred to herein as the “pichia host of the present invention.” In addition to the transformation of the pichia host by plasmids or vectors, the transformation of the chromosomes of the pichia host is also encompassed by the present invention. A strong (inducible) promoter (in place of the natural promoter of the natural accessory protein) may be introduced into the chromosomes of the pichia host; or another copy of the accessory protein gene sequence under the control of another (strong) promoter may be introduced into the chromosomes.

[0054] In another aspect, the present invention relates to a pichia host that expresses (or can express under appropriate conditions) the polypeptide of the present invention (where HAC1 splicing expression is enhanced); and / or contains the nucleic acid of the present invention and / or the gene construct of the present invention.

[0055] In a preferred embodiment, the Pichia host is Pichia pastris.

[0056] In another preferred embodiment, the Pichia pastris strain is selected from Pichia pastris X33 and Pichia pastris NRRL Y-11430.

[0057] The present invention further relates to methods for preparing the nucleic acids, gene constructs, and pichia hosts of the present invention; and to the use of the nucleic acids, gene constructs, and pichia hosts of the present invention for the production of immunoglobulin monovariate domains and polypeptides comprising them.

[0058] The present invention further relates to polypeptides and / or immunoglobulin monovariable domains that can be obtained by any of the methods described herein, pharmaceutical compositions and other compositions comprising such polypeptides and / or immunoglobulin monovariable domains, and therapeutic uses of such polypeptides and / or immunoglobulin monovariable domains, or treatment methods comprising the use of such polypeptides and / or immunoglobulin monovariable domains. [Brief explanation of the drawing]

[0059] [Figure 1] Nanobody A was expressed in two different pichia clones prepared as described in Example 1.2. One pichia clone contained one copy of the nanobody A expression cassette within its genome. The second clone contained more than one inserted copy of the nanobody A expression cassette within its genome. Equivolute supernatants from the different clones were compared on an SDS-PAGE gel. Concentration analysis was performed for relative quantification against the band corresponding to the intact nanobody product. Band volume quantification was performed using Imagequant software (GE Healthcare). An inverse correlation was observed between copy number and yield. [Figure 2] Clones transformed with nanobody A and accessory protein libraries were tested for improved nanobody A expression levels. Equivolute supernatants from different clones were compared on an SDS-PAGE gel. Concentration analysis was performed for relative quantification against the band corresponding to intact nanobody products. Band volume quantification was performed using Imagequant software (GE Healthcare). Compared to their corresponding reference clones without accessory proteins (copy number 1 and copy number > 1 of Ref), four clones (6H1, 4C2, 5A6, 9C4) were found to secrete significantly higher levels of nanobody A in a shaking flask. Ref: Reference clone with the indicated copy number of nanobody A expression cassette inserted into the genome. [Figure 3]Expression of reference clones with more than one copy of nanobody A expression cassettes transformed with one of the auxiliary proteins Kar2p, RPP0, Hac1 splice variant, or PDI1 in a shaking flask. Nanobody yield was analyzed on SDS-PAGE and compared to the nanobody yield from the reference clone (without auxiliary proteins). Concentration analysis was performed for relative quantification against the band corresponding to intact nanobody products. Band volume quantification was performed using Imagequant software (GE Healthcare).

[0060] Detailed description of the invention definition Unless otherwise indicated or defined, all terms used have their ordinary meanings in the art, which will be apparent to those skilled in the art. For example, see standard handbooks, e.g., Sambrook et al. 1989 (Molecular Cloning: A Laboratory Manual, 2 nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory Press), Ausubel et al. 1987 (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York), Lewin 1985 (Genes II, John Wiley & Sons, New York, NY), Old et al. 1981 (Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2) nd Ed., University of California Press, Berkeley, CA), Roitt et al. 2001 (Immunology, 6 thEd., Mosby / Elsevier, Edinburgh), Roitt et al. 2001 (Roitt's Essential Immunology, 10 th Ed., Blackwell Publishing, UK) and Janeway et al. 2005 (Immunobiology, 6 th See Ed., Garland Science Publishing / Churchill Livingstone, New York, and the general background technology cited herein.

[0061] Unless otherwise noted, all methods, processes, techniques, and operations not specifically described can and have been carried out in ways that are known to those skilled in the art. For example, refer here again to the standard handbooks and general background art described herein, as well as further literature cited therein; and to the following reviews describing, for example, techniques for protein engineering such as affinity maturation, and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins: Presta 2006 (Adv. Drug Deliv. Rev. 58: 640), Levin and Weiss 2006 (Mol. Biosyst. 2: 49), Irving et al. 2001 (J. Immunol. Methods 248: 31), Schmitz et al. 2000 (Placenta 21 Suppl. A: S106), and Gonzales et al. 2005 (Tumour Biol. 26: 31).

[0062] When a nucleotide sequence or amino acid sequence is said to "contain" another nucleotide sequence or amino acid sequence, or to "substantially consist of" another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence is incorporated into the first described nucleotide sequence or amino acid sequence, but usually this generally means that the first described nucleotide sequence or amino acid sequence contains, within its sequence, a nucleotide residue chain or amino acid residue chain having the same nucleotide sequence or amino acid sequence as the latter sequence, regardless of how the first described sequence was actually constructed or obtained (which is obtained, for example, by any suitable method described herein). Using a non-limiting example, when a polypeptide of the present invention is said to contain an immunoglobulin monovariate domain, this may mean that an immunoglobulin monovariate domain sequence is incorporated into the polypeptide sequence of the present invention, but usually this generally means that the polypeptide of the present invention contains, within its sequence, a sequence of an immunoglobulin monovariate domain, regardless of how the polypeptide of the present invention was constructed or obtained. Furthermore, when a nucleic acid sequence or nucleotide sequence is said to contain another nucleotide sequence, the first described nucleic acid sequence or nucleotide sequence is preferably such that, when expressed in an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of the expression product (in other words, the latter nucleotide sequence is within the same reading frame as the larger nucleic acid sequence or nucleotide sequence that was first described).

[0063] "Substantially" means that the immunoglobulin monovariate used in the method of the present invention is exactly the same as the polypeptide of the present invention, or corresponds to the polypeptide of the present invention having a small number of amino acid residues, for example 1 to 20 amino acid residues, for example 1 to 10 amino acid residues, preferably 1 to 6 amino acid residues, for example 1, 2, 3, 4, 5, or 6 amino acid residues, attached to the amino terminus, carboxy terminus, or both the amino terminus and carboxy terminus of the immunoglobulin monovariate domain.

[0064] A nucleic acid or amino acid is considered "(substantially) isolated" if, for example, it is separated from at least one other component typically present in the source or culture medium, such as another nucleic acid, another protein / polypeptide, another biological component or macromolecule, or at least one contaminant, impurity, or minor component, compared to the reaction medium or culture medium from which it was obtained. In particular, a nucleic acid or amino acid is considered "(substantially) isolated" if it has been purified at least twice, particularly at least ten times, more specifically at least 100 times, and up to 1000 times or more. A nucleic acid or amino acid that is "(substantially) isolated" is preferably substantially homogeneous as determined by appropriate techniques, such as appropriate chromatographic techniques, such as polyacrylamide gel electrophoresis.

[0065] The terms “expression” or “expressing” of a polypeptide, such as the immunoglobulin monovariable domain and / or polypeptide or accessory protein of the present invention, refer to the process by which information from a gene is used to synthesize a functional gene product (i.e., the immunoglobulin monovariable domain and / or polypeptide or accessory protein of the present invention). When it is said that an accessory protein or polypeptide, such as the immunoglobulin monovariable domain and / or polypeptide of the present invention, is “expressed from” a nucleic acid, gene construct, or chromosome, it means that it is synthesized through a process by which information from a nucleic acid, gene construct, or chromosome is used to synthesize a functional gene product (i.e., the immunoglobulin monovariable domain and / or polypeptide or accessory protein of the present invention). When it is said that a protein is “co-expressed,” it means that the protein is expressed simultaneously. To “enhance gene expression” means that the production of a gene product (e.g., an accessory protein) is increased compared to the production of a gene product without enhanced gene expression. As further described, the expression of a particular gene can be enhanced by a variety of means, including, for example, the use of appropriate regulatory sequences such as strong promoters, and / or increasing the amount of the gene by, for example, increasing the copy number of each gene.

[0066] As used in this invention, the term "yield" refers to the amount of the immunoglobulin monovariable domain and / or polypeptide of the present invention produced in a functional form when expressed in a Pichia host. Yield is expressed as grams (g) of the immunoglobulin monovariable domain and / or polypeptide of the present invention per liter (L) of culture medium.

[0067] Immunoglobulin monovariable domain Unless otherwise specified, the term “immunoglobulin sequence” as used herein to refer to a heavy chain antibody or a conventional quadruple chain antibody means a complete-sized antibody, its individual chains, and all parts thereof, its domains, or its fragments (antigen-binding domains or fragments, e.g., V, each). HHDomain or V H / V L It is used as a general term that includes both (but is not limited to these) domains. Further, as used herein (e.g., in terms such as "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "V HH sequence" or "protein sequence"), the term "sequence" should generally be understood to include both the relevant amino acid sequence, as well as the nucleic acid sequence or nucleotide sequence encoding it, unless the context requires a more limited interpretation.

[0068] The term "immunoglobulin single variable domain", which is used interchangeably with "single variable domain", is defined as a molecule in which the antigen-binding site is present on and formed by a single immunoglobulin domain. Thus, an immunoglobulin single variable domain is different from "conventional" immunoglobulins or their fragments (e.g., Fab, scFv, etc.) in which two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in a conventional immunoglobulin, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both VH and VL contribute to the antigen-binding site, i.e., a total of six CDRs would be involved in the formation of the antigen-binding site.

[0069] In contrast, the binding site of an immunoglobulin single variable domain is formed by a single VH domain or VL domain. Thus, the antigen-binding site of an immunoglobulin single variable domain is formed by three or fewer CDRs.

[0070] Thus, the terms "immunoglobulin single variable domain" and "single variable domain" do not include conventional immunoglobulins or their fragments that require the interaction of at least two variable domains for the formation of an antigen-binding site. However, these terms do include fragments of conventional immunoglobulins in which the antigen-binding site is formed by a single variable domain.

[0071] Generally, a single variable domain would be a substantial amino acid consisting of four framework regions (FR1-FR4, respectively) and three complementarity-determining regions (CDR1-CDR3, respectively); or any suitable fragment of such amino acids (which would typically contain at least some of the amino acid residues that form at least one of the CDRs). Such single variable domains and fragments are most preferably those that contain an immunoglobulin fold or are capable of forming an immunoglobulin fold under appropriate conditions. Thus, a single variable domain may include, for example, a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or VHH sequence) or a suitable fragment thereof; insofar as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit substantially consisting of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit, as is the case with variable domains present in conventional antibodies and scFv fragments, for example, those that need to interact with another variable domain to form a functional antigen-binding domain, for example, through a VH / VL interaction);

[0072] In one embodiment of the present invention, the immunoglobulin single variable domain is a light chain variable domain sequence (e.g., a VL sequence) or a heavy chain variable domain sequence (e.g., a VH sequence); more specifically, the immunoglobulin single variable domain may be a heavy chain variable domain sequence derived from a conventional quadruple-chain antibody, or a heavy chain variable domain sequence derived from a heavy chain antibody.

[0073] For example, a single variable domain or an immunoglobulin single variable domain (or an amino acid suitable for use as an immunoglobulin single variable domain) may be a (single) domain antibody (or an amino acid suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid suitable for use as a dAb), or a nanobody (such as those defined herein, including but not limited to VHH); another single variable domain, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, see also the prior art cited herein, as well as European Patent No. 0368684. For the term "dAb," see, for example, Ward et al. 1989 (Nature 341: 544), Holt et al. 2003 (Trends Biotechnol. 21: 484), as well as, for example, International Publication Nos. 04 / 068820, 06 / 030220, 06 / 003388, and other published patent applications of Domantis Ltd. It should also be noted that, although less desirable in the context of the present invention because they are not of mammalian origin, single variable domains may originate from specific species of sharks (e.g., the so-called "IgNAR domain," see, for example, International Publication No. 05 / 18629).

[0074] In particular, a single variable immunoglobulin domain may be a nanobody (as defined herein) or a suitable fragment thereof. [Note: Nanobody, nanobodies, and nanoclone are registered trademarks of Ablynx NV]. For a general description of nanobodies, see the prior art cited herein, for example, in International Publication No. 08 / 020079 (page 16).

[0075] The immunoglobulin sequence, particularly the amino acid sequence and structure of nanobodies, may be considered, but not limited to, to consist of four framework regions, or "FRs," referred to in the art and herein as "framework region 1," or "FR1"; "framework region 2," or "FR2"; "framework region 3," or "FR3"; and "framework region 4," or "FR4"; the framework regions are interrupted in the art by three complementarity-determining regions, or "CDRs," referred to in the art as "complementarity-determining region 1," or "CDR1"; "complementarity-determining region 2," or "CDR2"; and "complementarity-determining region 3," or "CDR3."

[0076] The total number of amino acid residues in a nanobody may be in the range of 110 to 120, preferably 112 to 115, and most preferably 113. However, portions, fragments, analogues, or derivatives of nanobody (as further described herein) are not particularly limited in terms of their length and / or size, as long as such portions, fragments, analogues, or derivatives satisfy the further requirements outlined herein and are also preferably suitable for the purposes described herein.

[0077] For further explanation of VHH and nanobodies, please refer to the review article by Muyldermans 2001 (Rev. Mol. Biotechnol. 74: 277), and the following patent application listed as general background technology: Vrije Universiteit International Publication Nos. 94 / 04678, 95 / 04079, and 96 / 34103 of Brussel; International Publication Nos. 94 / 25591, 99 / 37681, 00 / 40968, 00 / 43507, 00 / 65057, 01 / 40310, 01 / 44301, European Patent No. 1134231, and International Publication No. 02 / 48193 of Unilever; International Publication Nos. 94 / 25591, 99 / 37681, 00 / 40968, 00 / 43507, 00 / 65057, 01 / 40310, 01 / 44301, European Patent No. 1134231, and International Publication No. 02 / 48193; Vlaams Instituut voor Biotechnologie International Publication Nos. 97 / 49805, 01 / 21817, 03 / 035694, 03 / 054016, and 03 / 055527 of (VIB); International Publication No. 03 / 050531 of Algonomics NV and Ablynx NV; International Publication No. 01 / 90190 by the National Research Institute of Canada; International Publication No. 03 / 025020 (=European Patent No. 1433793) by the Institute of Antibodies; and Ablynx See International Publication Nos. 04 / 041867, 04 / 041862, 04 / 041865, 04 / 041863, 04 / 062551, 05 / 044858, 06 / 40153, 06 / 079372, 06 / 122786, 06 / 122787, and 06 / 122825 by NV, as well as other published patent applications by Ablynx NV. Furthermore, with reference to other prior art described in these applications, particularly the list of documents listed on pages 41-43 of the International Publication No. 06 / 040153 of the international application, this list and the documents are incorporated herein by reference.As described in these publications, nanobodies (particularly VHHs and partially humanized nanobodies) can be characterized in particular by the presence of one or more “characteristic residues” within one or more framework sequences. Further descriptions of nanobodies, including the humanization and / or camelization of nanobodies, as well as other modifications, parts or fragments, derivatives, or “nanobody fusions,” multivalent constructs (including examples of several non-limiting linker sequences), and various modifications to extend the half-life of nanobodies, and their preparations, can be found, for example, in International Publication No. 08 / 101985 and International Publication No. 08 / 142164.

[0078] Therefore, in the sense of the present invention, the terms “immunoglobulin monovariate domain” or “monovariate domain” include polypeptides derived from non-human sources, preferably camels, preferably camel heavy chain antibodies. These may be humanized as previously described. Furthermore, the terms also include polypeptides derived from non-camel sources, such as mice or humans, that have been “camelized,” as described, for example, in Davies and Riechmann 1994 (FEBS 339: 285), 1995 (Biotechonol. 13: 475), and 1996 (Prot. Eng. 9: 531) and Riechmann and Muyldermans 1999 (J. Immunol. Methods 231: 25).

[0079] The term “immunoglobulin monovariable domain” encompasses immunoglobulin sequences of various origins, including those from mouse, rat, rabbit, donkey, human, and camel. It also includes complete human immunoglobulin sequences, humanized immunoglobulin sequences, or chimeric immunoglobulin sequences. For example, it includes camel immunoglobulin sequences and humanized camel immunoglobulin sequences, or camelized immunoglobulin monovariable domains, such as camelized dAb as described by Ward et al (see, for example, International Publication No. 94 / 04678 and Davies and Riechmann 1994, 1995, and 1996).

[0080] The present invention may be used for the expression or production of any of the immunoglobulin monovariable domains described herein. The present invention may also be used for the expression or production of the polypeptide of the present invention (i.e., a polypeptide comprising or substantially comprising such an immunoglobulin monovariable domain). In particular embodiments, the present invention may be used for the expression or production of an immunoglobulin monovariable domain comprising two disulfide crosslinks. The present invention may also be used for the expression or production of the polypeptide of the present invention having two disulfide crosslinks (i.e., a polypeptide comprising or substantially comprising such an immunoglobulin monovariable domain). The present invention may also be used for the expression or production of the polypeptide of the present invention (i.e., a polypeptide comprising or substantially comprising such an immunoglobulin monovariable domain having two disulfide crosslinks).

[0081] All VHHs are known to contain at least one disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92 (see Kabat's patent application for Ablynx NV and Muyldermans and Lauwereys 1999, J. Mol. Recognit. 12: 131 for numbering). While most VHHs contain only this single disulfide bridge, some VHHs are known to contain a total of two (or, in exceptional cases, three) disulfide bridges. For example, classes of VHHs (and nanobodies) referred to as "VHH-1 type," "VHH-1 class," or otherwise (as further defined herein) generally have a second disulfide bridge between the cysteine ​​residue at position 50 in CDR2 and a cysteine ​​residue located in CDR3 (or, in exceptional cases, in CDR1 or CDR2). Furthermore, some VHHs derived from camels or dromedaries often have a disulfide bridge between a cysteine ​​residue located in CDR1 (or at position 45 in FR2) and a cysteine ​​residue located in CDR3 (Vu et al. 1997, Mol. Immunol. 34: 1121; Muyldermans and Lauwereys 1999). Some VHHs derived from llamas sometimes have a disulfide bridge between a cysteine ​​residue located in CDR1 (e.g., at position 33) and a cysteine ​​residue located in CDR3 (Vu et al., 1997).

[0082] In one specific but non-limiting embodiment, the immunoglobulin monovariable domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between a cysteine ​​residue in one of the framework regions and a cysteine ​​residue in one of the CDR regions.

[0083] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between a cysteine ​​residue in one of the framework regions and a cysteine ​​residue in CDR3.

[0084] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between the cysteine ​​residue in framework 2 (FR2) and the cysteine ​​residue in CDR3.

[0085] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between the cysteine ​​residue at position 45 in framework 2 (FR2) and the cysteine ​​residue in CDR3 (as in some VHHs derived from camels and dromedaries).

[0086] In another specific but non-limiting embodiment, the immunoglobulin monovariable domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between a cysteine ​​residue in one CDR and a cysteine ​​residue in another CDR.

[0087] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between a cysteine ​​residue in CDR3 and a cysteine ​​residue in another CDR (particularly in CDR1, such as some VHHs derived from camels, dromedaries, and llamas).

[0088] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between a cysteine ​​residue at position 22 and a cysteine ​​residue at position 92, and further includes a disulfide bridge formed between a cysteine ​​residue in CDR1 and a cysteine ​​residue in another CDR.

[0089] In another specific but non-limiting embodiment, the immunoglobulin monovariable domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between cysteine ​​residues within CDR1 and cysteine ​​residues within CDR1.

[0090] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between the cysteine ​​residue in CDR1 and the cysteine ​​residue in CDR3 (as in some VHHs derived from camels, dromedaries, and llamas).

[0091] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between the cysteine ​​at position 33 and the cysteine ​​residue within CDR3 (as in some VHHs derived from camels, dromedaries, and llamas).

[0092] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between a cysteine ​​residue at position 22 and a cysteine ​​residue at position 92, and further includes a disulfide bridge formed between a cysteine ​​residue in CDR2 and a cysteine ​​residue in another CDR.

[0093] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between cysteine ​​residues within CDR2 and cysteine ​​residues within CDR2.

[0094] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between the cysteine ​​residue in CDR2 and the cysteine ​​residue in CDR3 (like some VHHs derived from llama).

[0095] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between the cysteine ​​residue at position 50 and a cysteine ​​residue in another CDR, such as CDR1, CDR2, or CDR3 (as in the VHH and nanobody of the VHH1 type).

[0096] In another specific but non-limiting embodiment, the immunoglobulin monovariate domain includes a disulfide bridge between the cysteine ​​residue at position 22 and the cysteine ​​residue at position 92, and further includes a disulfide bridge formed between the cysteine ​​residue at position 50 and the cysteine ​​residue within CDR3 (as in the VHH1 type VHH and nanobody).

[0097] In preferred but non-limiting embodiments, the immunoglobulin monovariable domain may be a "VHH1 type immunoglobulin monovariable domain." For example, the amino acids in the immunoglobulin monovariable domain or polypeptide of the present invention may be such that the VHH1 type immunoglobulin monovariable domain or VHH1 type sequence is the VHH1 common sequence (SEQ ID NO: 46): [ka] A sequence is said to be a "VHH1 type immunoglobulin monovariate domain" or "VHH1 type sequence" if it has an 85% identity rate (using the blastp algorithm with standard settings, i.e., the bloom62 scoring matrix) and necessarily has a cysteine ​​at position 50, i.e., Cys50 (using Kabat numbering). These VHH1 type immunoglobulin monovariate domains generally have (or can form) a disulfide bridge between Cys50 and a cysteine ​​residue within CDR3 (or exceptionally within CDR1 or CDR2).

[0098] The amino acid sequence, for example, the immunoglobulin monovariable domain or polypeptide of the present invention, is a VHH2 type immunoglobulin monovariable domain or VHH2 type sequence, which is a VHH2 common sequence (SEQ ID NO: 47): [ka] A sequence is said to be a "VHH2 type immunoglobulin single variable domain" or "VHH2 type sequence" if it has an 85% identity rate (using the blastp algorithm with standard settings, i.e., the bloom62 scoring matrix).

[0099] The amino acid sequence, for example, the immunoglobulin monovariable domain or polypeptide according to the present invention, is a VHH3 type immunoglobulin monovariable domain or VHH3 type sequence, which is a VHH3 common sequence (SEQ ID NO: 48): [ka] A sequence is said to be a "VHH3 type immunoglobulin single variable domain" or "VHH3 type sequence" if it has an 85% identity rate (using the blastp algorithm with standard settings, i.e., the bloom62 scoring matrix).

[0100] While the present invention is particularly well suited to the expression of VHH1 (where the presence of two disulfide crosslinks is very common), it should be noted that the present invention can also be applied to the expression of VHH2 or VHH3 (which may or may not contain two disulfide crosslinks, but this is less common).

[0101] For a general description and for some non-limiting examples of nanobodies (and polypeptides containing them) that are directed toward c-Met and can be expressed / produced using the methods described herein, see International Publication No. 2012 / 042026 and International Publication No. 2013 / 045707.

[0102] For a general description and some non-limiting examples of nanobodies (and polypeptides containing them) that are directed toward TNF and can be expressed / produced using the methods described herein, see Ablynx NV's U.S. Provisional Application US62 / 254,375 (see also PCT / EP2016 / 077595).

[0103] The inventors also anticipate that this doctrine is not only particularly applicable to VHHs and nanobodies having two or more disulfide crosslinks, such as VHH-1, but also to other immunoglobulin monovariate domains containing two or more disulfide crosslinks (e.g., (mono)domain antibodies, dAbs, shark-derived IgNAR domains, etc.).

[0104] The polypeptide of the present invention The immunoglobulin monovariable domains prepared by the method of the present invention may comprise or substantially comprise one or more (at least one) immunoglobulin monovariable domains and may further comprise one or more additional amino acid sequences (all optionally linked via one or more suitable linkers), and may form part of a protein or polypeptide (hereinafter referred to as "polypeptide of the present invention"). The term "immunoglobulin monovariable domain" may also encompass such polypeptides of the present invention. One or more immunoglobulin monovariate domains may be used as binding units in such proteins or polypeptides, which may optionally contain one or more further amino acids capable of acting as binding units, thereby providing monovalent, polyvalent, or multispecific polypeptides of the present invention, respectively (for polyvalent and multispecific polypeptides containing one or more VHH domains and their preparation, see also Conrath et al. 2001 (J. Biol. Chem. 276: 7346) and, for example, International Publication Nos. 96 / 34103, 99 / 23221 and 2010 / 115998).

[0105] The polypeptide of the present invention may comprise or substantially consist of a single immunoglobulin monovariable domain, as outlined above. Such polypeptides are also referred to herein as monovalent polypeptides.

[0106] The polypeptides of the present invention may also include constructs comprising two or more antigen-binding sites in the form of a single variable domain, as outlined above. For example, by linking two (or more) immunoglobulin single variable domains having the same or different antigen specificity, constructs such as bivalent, trivalent, or polyvalent can be formed. By combining two or more immunoglobulin single variable domains having two or more specificities, constructs such as bispecific, tripspecific, can be formed. For example, the immunoglobulin single variable domains according to the present invention may include two or three immunoglobulin single variable domains directed toward the same target, or one or two immunoglobulin single variable domains directed toward target A and one immunoglobulin single variable domain directed toward target B. Such constructs and modifications thereof, which are readily conceivable to those skilled in the art, are all encompassed by the term polypeptide of the present invention as used herein.

[0107] Furthermore, the method of the present invention also prepares fused immunoglobulin sequences, including tags or other functional parts such as toxins, labels, or radiochemicals.

[0108] In another embodiment, the polypeptide of the present invention comprising or substantially comprising one or more immunoglobulin monovariable domains (or suitable fragments thereof) may further comprise one or more other groups, residues, moieties, or binding units. Such further groups, residues, moieties, binding units, or amino acid sequences may or may not provide further functionality to the immunoglobulin monovariable domain (and / or to the polypeptide in which it is present), and may or may not modify the properties of the immunoglobulin monovariable domain.

[0109] For example, such further groups, residues, moieties, or binding units may be one or more additional amino acids, and the compound, construct, or polypeptide is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting embodiment, the one or more other groups, residues, moieties, or binding units are immunoglobulins. More preferably, the one or more other groups, residues, moieties, or binding units are selected from the group consisting of domain antibodies, amino acids suitable for use as domain antibodies, single-domain antibodies, amino acids suitable for use as single-domain antibodies, "dAb", amino acids suitable for use as dAb, or nanobodies.

[0110] Alternatively, such groups, residues, moieties, or binding units may be chemical groups, residues, or moieties that are themselves biologically and / or pharmacologically active or not. For example, but not limited to, such groups may be linked to one or more immunoglobulin monovariable domains to provide "derivatives" of immunoglobulin monovariable domains.

[0111] In a preferred but non-limiting embodiment, the aforementioned further residues may be effective in preventing or reducing the binding of so-called "existing antibodies" to the polypeptide of the present invention. For this purpose, the polypeptides and constructs of the present invention may contain a C-terminal extension (X)n (wherein n is 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 (preferably 1 or 2, e.g., 1); each X is an independently selected (preferably native) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)), all of which are concurrently pending U.S. provisional applications, all of which are titled "Improved immunoglobulin variable domains": U.S. Patent No. 61 / 994552 filed on 16 May 2014; U.S. Patent No. 61 / 014,015 filed on 18 June 2014; U.S. Patent No. 62 / 040,167 filed on 21 August 2014; and U.S. Patent No. 62 / 047,560 filed on 8 September 2014 (all Ablynx See International Publication No. 2015 / 173325 of the international applications (belonging to NV) and the international applications published on November 19, 2015, based on these provisional applications.

[0112] Therefore, in the method of the present invention, the polypeptide may further include a C-terminal extension (X)n (where n is 1 to 5, for example 1, 2, 3, 4, or 5, and X is a natural amino acid, preferably no cysteine ​​at all).

[0113] In a preferred embodiment, the polypeptide expressed in the method of the present invention comprises or substantially comprises SEQ ID NO: 55. In a particular embodiment, such polypeptide comprises SEQ ID NO: 55.

[0114] In the polypeptide described above, one or more immunoglobulin monovariable domains and one or more groups, residues, moieties, or binding units may be linked to each other directly and / or via one or more appropriate linkers or spacers. For example, if one or more groups, residues, moieties, or binding units are amino acids, the linker may also be an amino acid, and the polypeptide thus obtained is a fusion protein or fusion polypeptide.

[0115] In one specific embodiment of the present invention, a polypeptide of the present invention having an extended half-life compared to a corresponding immunoglobulin monovariable domain is prepared. For example, a polypeptide of the present invention containing such a half-life-extending portion is a polypeptide in which an immunoglobulin monovariable domain is appropriately linked to one or more serum proteins or fragments thereof (e.g., (human) serum albumin or a suitable fragment thereof), or to one or more binding units capable of binding to a serum protein (e.g., a domain antibody, an amino acid suitable for use as a domain antibody, a single-domain antibody, an amino acid suitable for use as a single-domain antibody, "dAb", an amino acid suitable for use as a dAb, or serum albumin (e.g., human serum albumin), serum immunoglobulin (e.g., IgG), or Examples include: nanobodies capable of binding to serum proteins such as transferrin; polypeptides in which an immunoglobulin monovariable domain is linked to an Fc portion (e.g., human Fc) or an appropriate portion or fragment thereof; or polypeptides in which one or more immunoglobulin monovariable domains(s) are appropriately linked to one or more small proteins or peptides capable of binding to serum proteins (e.g., proteins and peptides described in International Publication No. 91 / 01743, International Publication No. 01 / 45746, or International Publication No. 02 / 076489, but not limited thereto).

[0116] Generally, polypeptides of the present invention having an extended half-life preferably have a half-life at least 1.5 times, preferably at least 2 times, for example at least 5 times, for example at least 10 times, or more than 20 times, that of the corresponding immunoglobulin monovariate domain or polypeptide of the present invention alone.

[0117] In preferred but non-limiting embodiments, such polypeptides of the present invention have a serum half-life of 1 hour or more, preferably 2 hours or more, more preferably 6 hours or more, for example 12 hours or more, or even 24, 48, or 72 hours or more, compared to the corresponding immunoglobulin monovariate domain or polypeptide of the present invention alone.

[0118] In another preferred but non-limiting embodiment, such polypeptides of the present invention exhibit a serum half-life in humans of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, and even more preferably at least 72 hours or more. For example, polypeptides of the present invention may have a half-life of at least 5 days (e.g., about 5 to 10 days), preferably at least 9 days (e.g., about 9 to 14 days), more preferably at least about 10 days (e.g., about 10 to 15 days), or at least about 11 days (e.g., about 11 to 16 days), more preferably at least about 12 days (e.g., about 12 to 18 days or more), or 14 days or more (e.g., about 14 to 19 days).

[0119] The method of the present invention is particularly suitable for immunoglobulin monovariable domains and / or polypeptides of the present invention that are not readily expressible in Pichia such as P. pastris, or that yield very low amounts when expressed under expression conditions applicable to use in these hosts, and therefore cannot be obtained in sufficient quantities when these immunoglobulin monovariable domains or polypeptides of the present invention are expressed in Pichia such as P. pastris. Accordingly, the method of the present invention is particularly suitable for immunoglobulin monovariable domains and / or polypeptides of the present invention that yield low amounts when expressed in a Pichia host (e.g., P. pastris) under standard Pichia expression conditions (as defined herein). As used in the present invention, "low yield" means that the yield of the immunoglobulin monovariable domain and / or polypeptide obtained is 0.5 g / L or less, for example, 0.4 g / L or less, 0.3 g / L or less, 0.2 g / L or less, 0.1 g / L or less, 0.05 g / L or less, 0.01 g / L or less, or even lower [expressed as grams (g) of the immunoglobulin monovariable domain or polypeptide of the present invention per liter (L) of culture medium].

[0120] The method of the present invention is also particularly suitable for immunoglobulin monovariable domains and polypeptides of the present invention, wherein the yield obtained when expressed in a pichia host (e.g., P. pastris) under standard pichia expression conditions (as defined herein) is inversely correlated (as defined herein) with the copy number of the nucleic acid encoding the immunoglobulin monovariable domain and / or polypeptide. A yield showing an "inverse correlation" means that the yield of the immunoglobulin monovariable domain and / or polypeptide obtained when the immunoglobulin monovariable domain and / or polypeptide is expressed in a pichia host having one copy of the nucleic acid encoding the immunoglobulin monovariable domain and / or polypeptide (under standard pichia expression conditions as defined herein) is higher than the yield of the immunoglobulin monovariable domain and / or polypeptide obtained when the immunoglobulin monovariable domain and / or polypeptide is expressed in a pichia host having more than one copy of the nucleic acid encoding the immunoglobulin monovariable domain and / or polypeptide (under standard pichia expression conditions as defined herein).

[0121] Preferred polypeptides for use in the method of the present invention include sequence numbers 49-54. These sequences also form separate embodiments of the present invention. Therefore, the present invention also relates to polypeptides having sequence numbers 49, 50, 51, 52, 53, 54, 55, or 56.

[0122] Other polypeptides particularly preferred for use in the method of the present invention include, or substantially consist of, an immunoglobulin monovariate domain comprising four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively) (where CDR1 is SEQ ID NO: 58, CDR2 is SEQ ID NO: 60, and CDR3 is SEQ ID NO: 62), for example, an immunoglobulin monovariate domain having the amino acid sequence of SEQ ID NO: 55 or 56.

[0123] Co-proteins As used herein, the term “auxiliary protein” refers to a protein that assists other molecular structures and / or proteins in performing their biological functions, but which itself does not appear in the structure of these other molecular structures and / or proteins when these other molecular structures and / or proteins perform their normal biological functions. Assistive proteins may, for example, modify the biophysical, pharmacological, and / or expression properties of other molecular structures and / or proteins. Assistive proteins may, but are not limited to, stabilize other molecular structures and / or proteins (e.g., through complex formation), modulate the activity of other molecular structures and / or proteins, increase the (surface) expression of other molecular structures and / or proteins, and / or assist in folding and / or association.

[0124] The auxiliary protein whose expression is enhanced by the method of the present invention is the functional HAC1 protein. The HAC1 protein may originate from any species, but is preferably of yeast origin, most preferably from Saccharomyces yeast, such as Saccharomyces, Komagataella, or Pichia (Hanzenula) yeast, such as Saccharomyces cerevisiae or Pichia pastris. In specific embodiments, the functional HAC1 protein is the "HAC1 splicing type" or "HAC1 splicing type protein" (both terms are used interchangeably herein). The HAC1 splicing type is the HAC1 protein obtained after a splicing event (removal of introns) on HAC1 mRNA, as described in Guerfal et al. 2010 (Microbial Cell Factories 9: 49). In more specific embodiments, the HAC1 splicing type protein is of Pichia origin. In a preferred embodiment, the HAC1 splicing protein has a sequence as described in Guerfal et al. 2010 (Microbial Cell Factories 9: 49; SEQ ID NO: 14).

[0125] In the method of the present invention, in addition to the HAC1 protein, the expression of one or more additional accessory proteins selected from protein disulfide isomerase (PDI1; EC5.3.4.1), Kar2p, and conserved ribosomal protein P0 (RPP0) is optionally enhanced. These accessory proteins may originate from any species, as long as their enhanced expression in the Pichia host yields an increased yield of the immunoglobulin monovariate domain and / or polypeptide of the present invention. In a preferred embodiment, the accessory proteins originate from fungi, such as yeast; preferably from yeast of the Saccharomyces genus, such as Saccharomyces, Chomagataera, or from yeast of the Pichia (Hanzenula) genus, such as Saccharomyces cerevisiae or Pichia pastris.

[0126] In a preferred embodiment, the auxiliary protein is: - P. Pastoris's HAC1 splicing type [ka] And, depending on the circumstances, one or more of the following: -P. pastris protein disulfide isomerase (PDI1): [ka] -P. Pastris's Kar2p: [ka] and - P. pastris 60S acidic ribosomal protein P0 (RPP0): [ka] Selected from.

[0127] Method of the present invention The present invention relates to a method for expressing and / or producing a monovariate immunoglobulin domain and / or a polypeptide containing the same in a Pichia host. The method of the present invention enhances the expression of HAC1 splicing protein in the Pichia host. The method of the present invention comprises the following steps: a) A step of expressing nucleic acids encoding the immunoglobulin monovariate domain and / or polypeptide of the present invention in a Pichia host; and b) A step of enhancing the expression of nucleic acids encoding HAC1 splicing proteins in the Pichia host; Depending on the circumstances, it may continue; c) A step of isolating and / or purifying the immunoglobulin single variable domain and / or polypeptide of the present invention obtained in this manner.

[0128] Therefore, the method of the present invention is a) A step of culturing the pichia host under conditions that allow the pichia host to proliferate; b) A step of maintaining the pichia host under conditions such that the pichia host expresses and / or produces the immunoglobulin monovariate domain and / or polypeptide of the present invention; and c) A step of enhancing the expression of nucleic acids encoding HAC1 splicing proteins in the Pichia host; Depending on the situation, continue: d) The step of isolating and / or purifying the immunoglobulin monovariate domain and / or polypeptide of the present invention obtained in this manner from the culture medium. Includes.

[0129] To produce / obtain expression of the immunoglobulin monovariate domain and / or polypeptide of the present invention, transformed pichia hosts can generally be retained, maintained, and / or cultured under conditions such as that the (desired) immunoglobulin monovariate domain and / or polypeptide of the present invention is expressed / produced. Suitable conditions will be obvious to those skilled in the art and will typically depend on the pichia host strain used, as well as regulatory sequences that control the expression of the (relevant) nucleotide sequence of the present invention.

[0130] Generally, suitable conditions include the use of a suitable culture medium, the presence of a suitable food source and / or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducer or compound (for example, when the nucleotide sequence(s) of the present invention are under the control of an inducible promoter); all of these can be selected by those skilled in the art. Here again, under such conditions, the immunoglobulin monovariate domain and / or polypeptide of the present invention may be expressed constitutively, transiently, or only when appropriately induced.

[0131] Culture conditions for the recombinant production of heterologous proteins in Pichia are, for example, described in Higgins and Cregg 1998 (Eds, Methods in Molecular Biology, Pichia protocols, Volume 103, 2) nd This has been described by Ed., Humana Press and by Invitrogen® (Invitrogen® Pichia Expression Kit; for recombinant protein expression in Pichia pastris; catalog number K1710-01). The production of immunoglobulin monovariate domains in P. pastris has been described extensively in International Publication Nos. 94 / 25591, 2010 / 125187, 2012 / 056000, and 2012 / 152823. The contents of these documents will be clearly referenced in relation to general culture techniques and methods, including appropriate media and conditions. The contents of these documents will be incorporated by reference. The present invention also relates to specific conditions described in the art, such as the general culture methods described in International Publication No. 94 / 25591, Gasser et al. 2006 (Biotechnol. Bioeng. 94: 535); Gasser et al. 2007 (Appl. Environ. Microbiol. 73: 6499), or Damasceno et al. 2007 (Microbiol. Biotechnol. 74: 381).

[0132] Pichia, particularly P. pastris, is typically cultured by fed-batch fermentation at 30°C using glycerol as the carbon source. Such media generally contain buffers, glycerol, trace elements, and ammonium hydroxide. Examples of buffers include (but are not limited to) H3PO4, CaSO4·2H2O, K2SO4, MgSO4·7H2O, and KOH. A typical growth medium (e.g., a basal salt medium) consists of 26.7 mL of 85% H3PO4, 0.93 g of CaSO4·2H2O, 18.2 g of K2SO4, 14.9 g of MgSO4·7H2O, 4.13 g of KOH, 40 g of glycerol, 2 mL of trace elements [6 g / L of copper sulfate·5H2O; 0.8 g / L of potassium iodide; 3 g / L of manganese sulfate·H2O; 0.2 g / L of sodium molybdate·2H2O; 0.2 g / L of boric acid; 0.5 g / L of copper sulfate; 20 g / L of zinc chloride; 65 g / L of iron sulfate·7H2O; 0.2 g / L of biotin; and 5 mL of concentrated sulfuric acid]. Ammonium hydroxide (NH4OH) is used to adjust the pH (e.g., pH 5) and as a nitrogen source. During the fed-batch culture period, glycerol (e.g., 50% v / v) is fed over several hours at a feed rate of, for example, 15 mL / L / h. The expression of the gene of interest encoding the desired immunoglobulin monovariate domain and / or polypeptide of the present invention is driven using the AOX1 promoter. Expression of the immunoglobulin monovariate domain and / or polypeptide of the present invention is carried out at 30°C with a methanol feed rate of 4–10 mL / L / h (e.g., 4 mL / L / h). These conditions are also referred to herein as “standard Pichia expression conditions”.

[0133] The expression of accessory proteins can be enhanced by commonly known means, including, for example, the use of appropriate regulatory sequences such as strong promoters, and / or increasing the amount of genes by, for example, increasing the copy number of each gene. The copy number can be increased, for example, by introducing a gene construct (plasmid or vector) suitable for the expression of the accessory protein. The presence of additional plasmids or vectors will increase the total copy number. Furthermore, gene constructs that can be amplified independently of the pichia host genome and exist in multiple copies within the pichia host may be used. For example, multicopy plasmids or vectors with 5 to 50 copy numbers may be present in the pichia host cells.

[0134] In addition to the transformation of the pichia host by plasmids or vectors, the transformation of the pichia host chromosome is also included in the present invention. A potent (inducible) promoter (in place of the natural promoter of the natural accessory protein) may be introduced into the host chromosome; or another copy of the accessory protein gene sequence under the control of another (potent) promoter may be introduced into the chromosome. Those skilled in the art know of many possibilities for enhancing the expression of the accessory protein, all of which are included in the present invention.

[0135] The accessory proteins(s) and the immunoglobulin monovariate domains and / or polypeptides of the present invention may be expressed from the same or different nucleic acids. Co-expression of two or more proteins may be achieved by the expression of two or more proteins on the same gene construct (integrated into a plasmid, vector, or host chromosome); or by the expression of two or more proteins on different gene constructs (integrated into a plasmid, vector, or host chromosome).

[0136] When expressed on the same gene construct, the nucleic acids encoding the two or more proteins are preferably located next to each other. The transcription of the nucleic acids encoding the two or more proteins may be controlled by a single promoter (located before both genes); or each nucleic acid encoding one of the two or more proteins may be controlled by separate promoters, which may be the same or different promoters.

[0137] When expressed from different gene constructs, the transcription of the nucleic acid encoding the polypeptide of the present invention and the transcription of the nucleic acid encoding one or more accessory proteins(s) may be controlled by two separate promoters, which may be the same or different.

[0138] The promoter may be either a constitutive promoter or an inductive promoter. In a preferred embodiment, the promoter is an inductive promoter.

[0139] The number of auxiliary proteins whose expression is enhanced by the method of the present invention may be one (HAC1 splicing protein), or it may be more than one, for example, two, three, four, five, or more. In a preferred embodiment, the number of auxiliary proteins whose expression is enhanced by the method of the present invention is one (HAC1 splicing protein).

[0140] In another preferred embodiment, the number of auxiliary proteins whose expression is enhanced by the method of the present invention is two, three, four, or even more. In this preferred embodiment, the expression of the HAC1 splicing protein is enhanced, and the expression of one or more additional auxiliary proteins is further enhanced. The additional auxiliary proteins may be any auxiliary proteins available and / or known in the art. Preferably, the additional auxiliary proteins are selected from PDI1, Kar2p, and RPP0.

[0141] Therefore, the method of the present invention also includes the expression and / or production of a single variable immunoglobulin domain and / or polypeptide containing the same in a Pichia host, and the expression of the HAC1 splicing protein and one or more additional accessory proteins selected from PDI1, Kar2p and RPP0 is enhanced. Therefore, the present invention also includes the following steps: a) A step of expressing a nucleotide sequence encoding the immunoglobulin monovariate domain and / or polypeptide of the present invention in a Pichia host; b) A step of enhancing the expression of nucleic acids encoding the HAC1 splicing protein and the expression of one, two, three (or more) nucleic acids encoding accessory proteins selected from PDI1, Kar2p, and RPP0 in the pichia host; Depending on the situation, continue: c) A step of isolating and / or purifying the immunoglobulin monovariable domain and / or polypeptide of the present invention obtained in this manner. This also relates to methods that include this.

[0142] In specific embodiments, the Method of the present invention is a) A step of culturing the pichia host under conditions that allow the pichia host to proliferate; b) A step of maintaining the pichia host under conditions such that the pichia host expresses and / or produces the immunoglobulin monovariate domain and / or polypeptide of the present invention; and c) A step of enhancing the expression of nucleic acids encoding the HAC1 splicing protein and the expression of one, two, three (or more) nucleic acids encoding auxiliary proteins selected from PDI1, Kar2p, and RPP0 in the Pichia host; Depending on the situation, continue: d) The step of isolating and / or purifying the immunoglobulin monovariate domain and / or polypeptide of the present invention obtained in this manner from the culture medium. Includes.

[0143] Therefore, in this particular embodiment of the method of the present invention, the following combinations of auxiliary proteins(s) may be enhanced: -PDI1 and HAC1 splicing type; -Kar2p and HAC1 splicing types; -RPP0 and HAC1 splicing type; -PDI1, Kar2p, and HAC1 splicing types; -PDI1, RPP0, and HAC1 splicing types; -Kar2p, RPP0 and HAC1 splicing types; and -PDI1, Kar2p, RPP0, and HAC1 splicing types.

[0144] If there are two or more accessory proteins, the accessory proteins may be expressed by the action of the same gene construct, such as the expression of two or more accessory proteins on a single plasmid or vector; or by the expression of two or more accessory proteins on different plasmids or vectors. In addition to the transformation of the pichia host by plasmids or vectors, the transformation of the pichia host chromosomes is also encompassed in this invention. A strong (inducible) promoter (in place of the native promoter of the native accessory protein) may be introduced into the pichia host chromosome; or another copy of the accessory protein gene sequence under the control of a different (strong) promoter may be introduced into the chromosome. When expressed on the same gene construct, two or more accessory proteins may be controlled by the same promoter or by different promoters. When expressed from different gene constructs, two or more accessory proteins may be controlled by two separate promoters, which may be the same or different. The promoters may be constitutive promoters or inducible promoters.

[0145] By using the above method, the inventors were able to increase the unexpectedly low yields sometimes observed with immunoglobulin monovariable domains and / or polypeptides containing them. Low yields were particularly observed with immunoglobulin monovariable domains (and / or polypeptides containing them) containing two disulfide crosslinks, immunoglobulin monovariable domains (and / or polypeptides containing them) that are VHH1 type immunoglobulin monovariable domains, and / or immunoglobulin monovariable domains (and / or polypeptides containing them) in which the yield obtained when expressed in a Pichia host under standard Pichia expression conditions was inversely correlated with the copy number of the nucleic acid encoding the immunoglobulin monovariable domain (and / or polypeptide).

[0146] Therefore, the present invention also provides a method for increasing the (expression and / or production) yield of immunoglobulin monovariate domains and / or polypeptides comprising them, which involves the following steps: a) A step of expressing nucleic acids encoding the immunoglobulin monovariate domain and / or polypeptide of the present invention in a Pichia host; and b) A step of enhancing the expression of nucleic acids encoding HAC1 splicing proteins in the pichia host; Depending on the situation, continue: c) A step of isolating and / or purifying the immunoglobulin monovariable domain and / or polypeptide of the present invention obtained in this manner. Includes.

[0147] Therefore, methods for increasing the (expression and / or production) yield of immunoglobulin monovariate domains and / or polypeptides are: a) A step of culturing the pichia host under conditions that allow the pichia host to proliferate; b) A step of maintaining the pichia host under conditions such that the pichia host expresses and / or produces the immunoglobulin monovariate domain and / or polypeptide of the present invention; and c) A step of enhancing the expression of nucleic acids encoding HAC1 splicing proteins in the Pichia host; Depending on the situation, continue: d) The step of isolating and / or purifying the immunoglobulin monovariate domain and / or polypeptide of the present invention obtained in this manner from the culture medium. Includes.

[0148] In a preferred embodiment, the method of the present invention provides a yield of the immunoglobulin monovariable domain and / or polypeptide of the present invention that is at least twice (preferably 3 times or 4 times or more, more preferably 5 times, 7.5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times or more) of the yield of the immunoglobulin monovariable domain and / or polypeptide of the present invention obtained by a method in which the expression of the HAC1 splicing protein is not enhanced.

[0149] Therefore, in a preferred embodiment, the present invention also provides a method for increasing the (expression and / or production) yield of immunoglobulin monovariate domains and / or polypeptides comprising them, which involves the following steps: a) A step of expressing nucleic acids encoding the immunoglobulin monovariate domain and / or polypeptide of the present invention in a Pichia host; and b) A step of enhancing the expression of nucleic acids encoding HAC1 splicing proteins in the pichia host; Depending on the situation, continue: c) A step of isolating and / or purifying the immunoglobulin monovariable domain and / or polypeptide of the present invention obtained in this manner. The method comprises and provides a yield of the immunoglobulin monovariable domain and / or polypeptide of the present invention that is at least twice (preferably 3 times or 4 times or more, more preferably 5 times, 7.5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times or more) of the yield of the immunoglobulin monovariable domain and / or polypeptide of the present invention obtained by a method in which the expression of the HAC1 splicing protein is not enhanced.

[0150] Therefore, methods for increasing the (expression and / or production) yield of immunoglobulin monovariate domains and / or polypeptides are: a) A step of culturing the pichia host under conditions that allow the pichia host to proliferate; b) A step of maintaining the pichia host under conditions such that the pichia host expresses and / or produces the immunoglobulin monovariate domain and / or polypeptide of the present invention; and c) A step of enhancing the expression of nucleic acids encoding HAC1 splicing proteins in the Pichia host; Depending on the situation, continue: d) The step of isolating and / or purifying the immunoglobulin monovariate domain and / or polypeptide of the present invention obtained in this manner from the culture medium. The method comprises and provides a yield of the immunoglobulin monovariable domain and / or polypeptide of the present invention that is at least twice (preferably 3 times or 4 times or more, more preferably 5 times, 7.5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times or more) of the yield of the immunoglobulin monovariable domain and / or polypeptide of the present invention obtained by a method in which the expression of the HAC1 splicing protein is not enhanced.

[0151] In another preferred embodiment, the method of the present invention provides a yield of the immunoglobulin monovariable domain and / or polypeptide of the present invention of 1 g / L or more, more preferably 1.5 g / L or more, 2 g / L or more, or even more than 2.5 g / L or more.

[0152] Therefore, in another preferred embodiment, the present invention also provides a method for increasing the (expression and / or production) yield of immunoglobulin monovariate domains and / or polypeptides comprising them, which involves the following steps: a) A step of expressing nucleic acids encoding the immunoglobulin monovariate domain and / or polypeptide of the present invention in a Pichia host; and b) A step of enhancing the expression of nucleic acids encoding HAC1 splicing proteins in the pichia host; Depending on the situation, continue: c) A step of isolating and / or purifying the immunoglobulin monovariable domain and / or polypeptide of the present invention obtained in this manner. The method provides a yield of the immunoglobulin monovariable domain and / or polypeptide of the present invention, comprising 1 g / L or more, more preferably 1.5 g / L or more, 2 g / L or more, or even more than 2.5 g / L or more.

[0153] Therefore, methods for increasing the (expression and / or production) yield of immunoglobulin monovariate domains and / or polypeptides are: a) A step of culturing the pichia host under conditions that allow the pichia host to proliferate; b) A step of maintaining the pichia host under conditions such that the pichia host expresses and / or produces the immunoglobulin monovariate domain and / or polypeptide of the present invention; and c) A step of enhancing the expression of nucleic acids encoding HAC1 splicing proteins in the Pichia host; Depending on the situation, continue: d) The step of isolating and / or purifying the immunoglobulin monovariate domain and / or polypeptide of the present invention obtained in this manner from the culture medium. The method provides a yield of the immunoglobulin monovariable domain and / or polypeptide of the present invention, comprising 1 g / L or more, more preferably 1.5 g / L or more, 2 g / L or more, or even more than 2.5 g / L or more.

[0154] The immunoglobulin monovariate domain and / or polypeptide of the present invention is produced extracellularly and isolated from the culture medium in which Pichia host cells are cultured.

[0155] Typically, but not necessarily, the immunoglobulin monovariable domain and / or polypeptide of the present invention will have at least one transport signal that directs the immunoglobulin monovariable domain and / or polypeptide of the present invention toward the peripheral material. In the present invention, the pichia host can be removed from the culture medium by routine means. For example, the pichia host can be removed by centrifugation or filtration. The solution obtained by removing the pichia host from the culture medium is also called the culture supernatant or clarified culture supernatant.

[0156] Furthermore, it will be apparent to those skilled in the art that the immunoglobulin monovariable domain and / or polypeptide of the present invention may be (first) produced in an immature form (as described above) and then subjected to post-translational modification depending on the Pichia host used. The immunoglobulin monovariable domain and / or polypeptide of the present invention may also be glycosylated, again depending on the Pichia host cell used.

[0157] The immunoglobulin monovariable domain and / or polypeptide of the present invention can subsequently be isolated from a pichia host and / or from the culture medium in which the pichia host was cultured by standard methods. Standard methods include, but are not limited to, chromatographic methods such as size exclusion chromatography, hydrophobic chromatography, ion exchange chromatography, and affinity chromatography. These methods can be carried out alone or in combination with other purification methods such as fractional precipitation techniques, gel electrophoresis, affinity techniques (e.g., using a specific and cleavable amino acid sequence fused to the immunoglobulin monovariable domain and / or polypeptide of the present invention) and / or immunological preparative techniques (i.e., using an antibody against the immunoglobulin monovariable domain and / or polypeptide of the present invention to be isolated). Those skilled in the art can, based on general knowledge, devise appropriate combinations of purification methods for the immunoglobulin monovariable domain. For specific examples, see the techniques cited herein.

[0158] Immunoglobulin monovariate domains and / or polypeptides containing them can be purified from the culture supernatant by a combination of protein A affinity chromatography, ion exchange chromatography, and size exclusion chromatography. Any reference to “purification steps” includes, but is not limited to, these specific methods. More specifically, immunoglobulin monovariate domains and / or polypeptides containing them can be purified from the culture supernatant using a process in which the clarified supernatant (obtained by centrifugation) is captured on a protein A resin; and subsequently subjected to a SOURCE15S (GE Healthcare) cation exchange chromatography step and a Superdex 75 (GE Healthcare) size exclusion chromatography step.

[0159] After removal of the Pichia host, the immunoglobulin monovariable domain and / or polypeptide of the present invention can be present in a wide variety of suitable buffers. Examples include, but are not limited to, PBS, Tris-HCl, histidine, or phosphate buffer. Alternatively, the immunoglobulin monovariable domain and / or polypeptide of the present invention may be present in physiological saline.

[0160] Generally, for pharmaceutical use, the immunoglobulin monovariable domains and / or polypeptides of the present invention may be formulated as pharmaceutical preparations or pharmaceutical compositions comprising at least one immunoglobulin monovariable domain and / or polypeptide of the present invention, at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more further pharmaceutically active polypeptides and / or compounds. Using non-limiting examples, such formulations may be in dosage forms suitable for oral administration, parenteral administration (e.g., by intravenous injection, intramuscular injection or subcutaneous injection or intravenous infusion), topical administration, inhalation, skin patch, implantation, suppository administration, etc. Such suitable dosage forms (which may be solid, semi-solid or liquid depending on the method of administration) and methods and carriers for use in their preparation will be apparent to those skilled in the art.

[0161] Nucleic acids and gene constructs of the present invention The present invention also relates to the immunoglobulin monovariate domain and / or polypeptide and nucleic acids encoding the HAC1 splicing type. These nucleic acids are also referred to herein as “the nucleic acids of the present invention.”

[0162] As will be apparent to those skilled in the art, the nucleic acids of the present invention may be in the form of a gene construct, may be present in a gene construct, and / or may be part of a gene construct. Such a gene construct generally comprises at least one nucleic acid of the present invention, optionally ligated to one or more sequences of gene constructs known in themselves, such as one or more suitable regulatory sequences (e.g., suitable promoters, enhancers, termination factors, etc.) and other sequences of gene constructs referred to herein. Such a gene construct comprising at least one nucleic acid of the present invention is also referred to herein as “gene constructs of the present invention.” Accordingly, the gene constructs of the present invention encode at least the immunoglobulin monovariable domain and / or polypeptide and HAC1 splicing type of the present invention.

[0163] The number of auxiliary proteins encoded by the nucleic acids(group) and gene constructs(group) of the present invention may be one (i.e., a HAC1 splicing protein) or more than one, for example, two, three, four, five, or more. In a preferred embodiment, the number of auxiliary proteins encoded by the nucleic acids and gene constructs(group) of the present invention is one (i.e., a HAC1 splicing protein). In another preferred embodiment, the number of auxiliary proteins encoded by the nucleic acids(group) and gene constructs(group) of the present invention is two or more.

[0164] Accordingly, the present invention also encompasses nucleic acids or gene constructs encoding the immunoglobulin single variable domain and / or polypeptide of the present invention and two or more accessory proteins (including HAC1 splicing proteins).

[0165] As discussed above, the immunoglobulin monovariable domains and / or polypeptides and auxiliary proteins(s) of the present invention may be co-expressed from a single nucleic acid and / or gene construct; or from different (separate) nucleic acids and / or gene constructs (possibly including expression from host chromosomes). All these nucleic acids and / or gene constructs encoding the immunoglobulin monovariable domains and / or polypeptides and / or auxiliary proteins(s) of the present invention (as a single construct or as separate constructs) are encompassed by the terms “nucleic acids(s) of the present invention” and “gene constructs(s) of the present invention.”

[0166] The nucleic acids of the present invention may be in the form of single-stranded or double-stranded DNA or RNA, and are preferably in the form of double-stranded DNA. For example, the nucleic acids of the present invention may be genomic DNA, cDNA, or synthetic DNA (for example, DNA having a codon usage frequency particularly adapted for expression in Pichia host cells).

[0167] According to one embodiment of the present invention, the nucleic acid of the present invention is in a substantially isolated form as defined herein. The nucleic acid of the present invention may also be in the form of a vector, such as a plasmid, cosmid, or YAC, or may be present within and / or part of such vector, which may also be in a substantially isolated form.

[0168] The nucleic acids of the present invention can be prepared or obtained by methods known in themselves, based on information regarding the immunoglobulin monovariable domain and / or polypeptide to be expressed and the auxiliary proteins(s) used for co-expression. Furthermore, as will be apparent to those skilled in the art, to prepare the nucleic acids of the present invention, several nucleotide sequences may be appropriately linked together, for example, at least one nucleotide sequence encoding the immunoglobulin monovariable domain and / or polypeptide of the present invention with at least one nucleotide sequence encoding an auxiliary protein.

[0169] The techniques for generating nucleic acids of the present invention will be apparent to those skilled in the art, and include, for example, automated DNA synthesis, site-directed mutagenesis, combination of two or more native and / or synthetic sequences (or two or more parts thereof), introduction of mutations resulting in the expression of truncated expression products, introduction of one or more restriction enzyme sites (e.g., to create cassettes and / or regions that can be readily digested and / or ligated using appropriate restriction enzymes), and / or introduction of mutations using PCR reactions with one or more "mismatch" primers. These and other techniques will be apparent to those skilled in the art, and here again, refer to standard handbooks, such as Sambrook et al. and Ausubel et al. described herein and the following examples.

[0170] The gene construct of the present invention may be DNA or RNA, preferably double-stranded DNA. The gene construct of the present invention may also be in a form suitable for transformation of a Pichia host, in a form suitable for integration into the genomic DNA of a Pichia host cell, or in a form suitable for independent replication, maintenance, and / or inheritance in a Pichia host. For example, the gene construct of the present invention may be in the form of a vector, such as a plasmid, YAC, viral vector, or transposon. In particular, the vector may be an expression vector, i.e., a vector capable of giving expression in a Pichia host.

[0171] In preferred but non-limiting embodiments, the gene construct of the present invention is a) At least one nucleic acid of the present invention operably connected to b) below, b) One or more regulatory sequences, e.g., a promoter and optionally an appropriate termination factor; And in some cases, also c) One or more further sequences of a gene construct that is known in itself The terms “control sequence,” “promoter,” “termination factor,” and “operably connected” have their usual meanings in the art (as further described herein); the “further sequences” present in the gene construct may be, for example, 3'- or 5'-UTR sequences, reader sequences, selection markers, expression marker / reporter genes, and / or sequences that can promote or increase transformation or integration (in efficiency). These sequences and other suitable sequences for such gene constructs will be apparent to those skilled in the art, and this depends, for example, on the type of construct used, the Pichia host strain, the manner in which the nucleotide sequence of the present invention of interest is to be expressed (e.g., via constitutive, transient, or inductive expression), and / or the transformation technique to be used. For example, control sequences, promoters, and termination factors known by themselves for the expression and production of antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and ScFv fragments) may be used in substantially similar manner.

[0172] Preferably, in the gene construct of the present invention, the at least one nucleic acid of the present invention and the regulatory sequence, and optionally one or more further sequences, are “operably linked” to one another, which generally means that they are functionally related to one another. For example, a promoter is considered to be “operably linked” to a coding sequence if the promoter can initiate or otherwise control / regulate the transcription and / or expression of the coding sequence (the coding sequence should be understood to be “under the control” of the promoter). Generally, when two nucleotide sequences are operably linked, they are in the same direction and usually within the same reading frame. They are also usually substantially continuous, but this is also not required. In one aspect of the present invention, a nucleotide sequence encoding an immunoglobulin monovariable domain and / or polypeptide of the present invention is operably linked to a nucleotide sequence encoding an accessory protein(s). They may be under the control of the same promoter, or they may each be under the control of separate (the same or different) promoters.

[0173] Methods for designing, preparing, or obtaining nucleic acid sequences for expression, constructing appropriate vectors, inserting nucleic acid sequences into the vectors, selecting appropriate Pichia host strains, introducing the vectors into the Pichia host strains, inducing or enabling the expression of polypeptides or proteins, isolating nucleic acids from the Pichia host strains, or identifying nucleic acid sequences and corresponding protein sequences are standard methods well known to anyone skilled in the art (Sambrook et al. 1989). Those skilled in the art can also, based on general knowledge, devise gene constructs suitable for expressing the immunoglobulin monovariate domain and / or polypeptide of the present invention in Pichia hosts. The present invention also refers to gene constructs described in the art, such as plasmids, promoters, and leader sequences described in International Publication No. 94 / 25591, Cereghino and Cregg 2000 (Curr. Opinion Biotechnol. 10: 422), Gasser et al. 2006 (Biotechnol. Bioeng. 94: 535), Gasser et al. 2007 (Appl. Environ. Microbiol. 73: 6499), or Damasceno et al. 2007 (Microbiol. Biotechnol. 74: 381).

[0174] Preferably, the regulatory sequences and further sequences of the gene construct of the present invention are such that they can impart their intended biological function in the Pichia host.

[0175] For example, a promoter, enhancer, or termination factor should be "operable" in the Pichia host, meaning that (for example) the promoter should be able to initiate or otherwise control / regulate the transcription and / or expression of the nucleotide sequence (as defined herein) to which it is operably linked, such as a coding sequence.

[0176] Some preferred but non-limiting examples of suitable promoters, termination factors, and further sequences that can be used for expression in a Pichia host; in particular, those described herein and / or used in the following examples.

[0177] Some particularly preferred promoters include promoters known for expression in Pichia hosts; in particular, those described herein and / or used in the examples. The specific sequence of a promoter determines its strength ("strong promoters" result in high transcription initiation rates). When it is said that the expression of a nucleic acid encoding a co-protein is controlled by a "strong promoter," it means that the expression of the nucleic acid encoding the co-protein is controlled by a promoter that results in a higher transcription initiation rate than the innate promoter that controls the transcription initiation of the innate co-protein. In addition to transcription-"promoting" sequences, promoters may include additional sequences known as operators that control the promoter's strength. For example, a promoter may include a protein binding site that induces or inhibits the binding of RNA to the promoter. The presence or absence of this protein will affect the promoter's strength. Such promoters are known as regulatory promoters.

[0178] The alcohol oxidase I (AOX1) promoter in P. pastris is one of the most potent and well-regulated promoters known. Conversely, the second alcohol oxidase (AOX2) in P. pastris is controlled by a much weaker promoter. The glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in P. pastris gives constitutively high levels of expression on glucose, glycerol, and methanol media (Waterham et al. 1997, Gene 186: 37). The formylaldehyde dehydrogenase (FLD1) promoter in P. pastris can be induced by either methanol or methylamine, and its expression levels are comparable to those obtained using the AOX1 promoter in methanol (Shen et al. 1998, Gene 216: 93). The peroxin 8 (PEX8) promoter yields low expression on glucose and is moderately induced (about 10-fold) when cells are converted to methanol (Johnson et al. 1999, Genetics 151: 1379).

[0179] The potent promoters in *H. polymorpha* include sequences derived from the methanol oxidase gene (MOX), formate dehydrogenase gene (FMD), and dihydroxyacetone synthase gene (DHAS) (Song et al. 2006, Biotechnol. Lett. 25: 1999). In *H. polymorpha*, the glyceraldehyde-3-phosphate dehydrogenase (GAP1) promoter (Sohn et al. 1999, Appl. Microbiol. Biotechnol. 51: 800) and the PMA1 promoter (Cox et al. 2000, Yeast 16: 1191) are constitutive sequences. The PMA1 promoter competes with the MOX promoter due to its high expression level.

[0180] The alcohol oxidase (AUG1) promoters P(MOD1) and P(MOD2) of Pichia methanolica are strongly and firmly regulated by methanol (P(MOD1) and P(MOD2)) and glycerol (P(MOD1) only) (Nakagawa et al. 2006, Yeast 23: 15).

[0181] Potent promoters derived from Candida boidinii include the alcohol oxidase (AOD1) promoter and the dihydroxyacetone synthase (DAS1) promoter (Yurimoto et al. 2000, Biochim. Biophys. Acta 1493: 56). Both the DAS1 promoter and the formate dehydrogenase (FMD) promoter are available for Candida boidinii (Sakai et al. 1995, Appl. Microbiol. Biotechnol. 42: 860; 1996, Biochim. Biophys. Acta 1308: 81) and Hanzenula polymorpha (Hollenberg and Gellissen 1997, Curr. Opin. Biotechnol. 8: 554).

[0182] The selection marker should be such that it enables the distinction, under appropriate selection conditions, between pichia host cells that have been (successfully) transformed with the nucleic acids of the present invention and pichia host cells that have not been (successfully) transformed. Some preferred but non-limiting examples of such markers are genes that confer resistance to antibiotics (e.g., zeosin, blastosidine, genethecin (G418), phleomycin, kanamycin, or ampicillin), genes that confer resistance to temperature, or genes that enable the pichia host to be maintained in the absence of certain factors, compounds, and / or (food) components in the culture medium that are essential for the survival of untransformed cells.

[0183] The leader sequence should be such that, in the pichia host, it enables the desired post-translational modification and / or secretion of the expression product from the cell. Therefore, the leader sequence may be any pro-sequence, pre-sequence, or pre-pro-sequence that is operational in the pichia host. However, typically, but not necessarily, the immunoglobulin monovariable domain and / or polypeptide of the present invention will have at least one transport signal that directs the immunoglobulin monovariable domain and / or protein of the present invention to the peripheral material. For example, leader sequences known in themselves for the expression and production of antibodies and antibody fragments (including, but not limited to, mono-domain antibodies and ScFv fragments) may be used in substantially the same manner.

[0184] Some preferred but non-limiting secretory sequences include the α-conjugation factor signal sequence from Saccharomyces cerevisiae, the acid phosphatase (PHO1) signal sequence from P. pastris, the phosphatase (pho1) reader sequence from P. pastris, the yeast invertase secretory signal (Suc), the human serum albumin signal peptide, the GAM1 signal sequence from Schwaniomyces occidentalis, and the hyperglycemic hormone (CHH) sequence from the European green crab (Carcinus maenas).

[0185] Those skilled in the art may also consider the use of predicted signal peptides derived from genome sequencing experiments. These predicted signal peptide sequences may originate from any species, but are preferably yeast-derived, most preferably from Saccharomyces yeast, e.g., Saccharomyces, Chomagataera, or Pichia (Hanzenula) yeast, e.g., Saccharomyces cerevisiae or Pichia pastris. Several preferred but non-limiting predicted signal peptides from P. pastris are described in De Schutter et al. 2009 (Nature Biotech 27(6): 561-566). For further information on the use of such predicted signal peptides for the generation of immunoglobulin monovariable domains, see International Publication No. 2012 / 152823.

[0186] Furthermore, known or predicted secretory sequences may be modified to improve the properties of the immunoglobulin monovariable domain and / or polypeptide produced by the present invention. Such modifications can improve the purity of the immunoglobulin monovariable domain and / or polypeptide by improving processing efficiency, for example. Modifications of the α-conjugation factor signal sequence for improving processing efficiency are described, for example, in International Publication No. 2012 / 152823.

[0187] The expression marker or reporter gene should be such that, in the Pichia host, it enables the detection of the expression of a gene construct (a gene sequence or nucleotide sequence present thereon). Such a reporter gene may also be expressed as a protein fusion with the immunoglobulin monovariate domain and / or polypeptide of the present invention. Some preferred but non-limiting examples include fluorescent proteins, e.g., GFP and luciferase LUC.

[0188] The gene constructs of the present invention can generally be provided by appropriately ligating the nucleic acids and / or nucleotide sequences(s) of the present invention to one or more of the above-mentioned further sequences using techniques described in general handbooks such as those by Sambrook et al. and Ausubel et al.

[0189] Often, the gene constructs of the present invention will be obtained by inserting the nucleic acid sequence or nucleotide sequence of the present invention into a suitable (expression) vector that is known in itself. Some preferred but non-limiting examples of suitable expression vectors are those used in the following examples and those described herein.

[0190] Some preferred but non-limiting vectors for use in the gene constructs of the present invention include vectors for expression in yeast or other fungal cells, e.g., pYES2 (Invitrogen), pUR3515 and pUR3501 (Sierkstra et al. 1991, Curr. Genet. 19: 81), and pichia expression vectors, e.g. (but not limited to) pPICZ vector for expression in P. pastris, pPIC3.5, pPIC3.5K, pPIC6a, pPIC9, pPIC9K, pHIL-D2, pHIL-S1, and pMET and pMETα for expression in P. metanolica, provided by Invitrogen. For a non-exclusive list of pichia expression vectors, see Daly and Hearn 2004 (J. Mol. Recognition 18: 119), Pichia Protocol 2007 (Ed. Cregg, 2 nd See also Ed., Humana Press, NJ and Gelissen 2000 (Appl. Microbiol. Biotechnol. 54: 741).

[0191] The present invention also includes a method for preparing nucleic acids and gene constructs of the present invention, comprising the step of cloning a nucleotide sequence encoding an auxiliary protein gene(s) and / or the immunoglobulin monovariate domain and / or polypeptide of the present invention into a suitable vector.

[0192] Pichia hosts can be transformed using the nucleic acids and / or gene constructs of the present invention, i.e., for the expression and / or production of the immunoglobulin monovariate domain and / or polypeptide of the present invention. Suitable techniques for transforming Pichia hosts will be apparent to those skilled in the art. Here again, refer to the above-mentioned handbook and patent application. For more details regarding the transformation procedure, see Invitrogen's EasySelect™ Pichia Expression Manual, Wu and Letchworth 2004 (BioTechniques 36: 152), and Pichia Protocol 2007 (Ed. Cregg, 2007). nd See Ed., Humana Press, NJ and Faber 1994 (Curr. Genet. 25: 305). After transformation, steps may be taken to detect and select Pichia host cells that have been successfully transformed using the nucleotide sequence / gene construct of the present invention. This may include, for example, a selection step based on selection markers present in the gene construct of the present invention, or a step including detection of the amino acid sequence of the present invention using, for example, a specific antibody.

[0193] Accordingly, the present invention further relates to the preparation of pichia host cells comprising the gene construct or nucleic acid of the present invention. Those skilled in the art can introduce the nucleic acid or gene construct of the present invention into a pichia host by routine measures, for example, by transformation. Then, those skilled in the art can select suitable pichia host cells comprising the nucleic acid or gene construct by, for example, monitoring the expression of the accessory protein at the nucleic acid level and / or protein level. A strain having a satisfactory expression level will be selected. High expression of the accessory protein is desirable, but it should not be so high as to compete with the expression of the immunoglobulin monovariate domain and / or polypeptide of the present invention. This can be determined by routine methods.

[0194] Transformed Pichia host cells (which may be in the form of a stable cell line) form a further embodiment of the present invention.

[0195] Pichia host Therefore, the present invention also relates to a Pichia host containing such a gene construct or nucleic acid as described above. The terms “Pichia host” and “Pichia host cell” are used interchangeably and refer to yeasts of the genus Pichia (Hanzenula and Hyphopichia are obsolete synonyms) of the family Saccharomycesaceae, having spherical, elliptical, or rectangular acuminate cells. Pichia are teleomorphs that form cap-shaped, hemispherical, or circular ascospores during sexual reproduction. The asexual generation of some Pichia species is Candida species. Asexual reproduction is by polylateral budding.

[0196] The present invention relates to, but is not limited to, Pichia hosts, which are suitable for the production of immunoglobulin monovariate domains and / or polypeptides containing them. For the purposes of the present invention, the term “Pichia host” also includes Hansenula and Candida species.

[0197] The Pichia host of the present invention may produce the immunoglobulin monovariate domain and / or polypeptide and HAC1 splicing protein of the present invention, and optionally one or more additional accessory proteins, such as protein disulfide isomerase (PDI1), Kar2p, or conserved ribosomal protein P0 (RPP0). It will typically be genetically modified to contain one or more nucleic acids encoding the immunoglobulin monovariate domain and / or polypeptide of the present invention, and to enhance the expression of the HAC1 splicing protein. Non-limiting examples of genetic modification include, for example, transformation using plasmids or vectors, or transduction using viral vectors. Some hosts may be genetically modified by fusion techniques. Genetic modifications include the introduction of separate nucleic acid molecules into the host, such as plasmids or vectors, and direct modification of the host's genetic material, such as by homologous recombination, for example, by integration into the host's chromosomes. Often, both combinations occur, for example, when the host is transformed using a plasmid, this plasmid will be integrated (at least partially) into the host's chromosomes by homologous recombination. Those skilled in the art know of appropriate methods for genetically modifying a host to enable the host to produce the immunoglobulin monovariable domain and / or polypeptide of the present invention.

[0198] Suitable Pichia hosts are obvious to those skilled in the art and may include, but are not limited to, yeasts such as Pichia, Hansenula, or Candida, e.g., metanotrof yeast, e.g., Pichia pastris, Pichia metanolica, Hansenula polymorpha (Pichia angusta), and Candida boidini. For a non-exclusive list of Pichia strains, see, for example, Gelissen 2000 (Appl. Microbiol. Biotechno. 54: 741). For P. pastris strains, though not limited to those listed, see Daly and Hearn 2004 (J. Mol. Recognition, 18: 119) and Pichia Protocol 2007 (Ed. Cregg, 2 nd Listed in Ed., Humana Press, NJ. Examples of P. pastris strains include (but are not limited to) X33, GS115, KM71, KM71H, SMD1163, SMD1165, SMD1168, SMD1168H, NRRL-Y11430, and GS200, provided by Invitrogen and described by Cereghino and Cregg 2004 (FEMS Microbiol. Rev. 24: 45), Macauley-Patrick et al. 2005 (Yeast 22: 249), and / or Damasceno et al. 2007 (Appl. Microbiol. Biotechno. 74: 381).

[0199] Examples of Hanzenula polymorpha strains include (but are not limited to) A16 (Veale et al. 1992, Yeast 8: 361), GF16 (Faber 1994, Proc. Natl. Acad. Sci. USA 91: 12985), CBS4732 (CCY38-22-2; ATCC34438, NRRL-Y-5445), DL-1 (NRRL-Y-7560; ATCC26012), and NCYC495 strain (CBS1976; ATAA14754, NRLL-Y-1798).

[0200] Examples of P. metanolica strains include (but are not limited to) PMAD11 and PMAD16 provided by Invitrogen. P. metanolica strains (IAM12901 and IAM12481) and C. boidini strain (IAM12875) were described by Nakagawa et al. 1996 (J. Fermentation Bioeng. 81: 498).

[0201] Furthermore, this specification refers to the general background art cited above, as well as, for example, International Publication No. 94 / 29457, Frenken et al. 1998 (Res. Immunol. 149: 589), van der Linden 2000 (J. Biotechnol. 80: 261), Joosten et al. 2003 (Microb. Cell Fact. 2: 1), and other references cited herein.

[0202] For industrial-scale production, a preferred heterologous host for the (industrial) production of immunoglobulin monovariate domain-containing protein therapeutics is the Pichia pastris strain, which is suitable for large-scale expression / production / fermentation, particularly for large-scale drug expression / production / fermentation. Suitable examples of such strains will be obvious to those skilled in the art. Such strains and production / expression systems are also available from companies such as Abysia Biologics (Billingham, North East England, UK), Biomeeva GmbH (Heidelberg, Germany), PharmedArtis GmbH (Aachen, Germany), Richter-Helm GmbH (Hamburg, Germany), and CMC Biologics GmbH (Copenhagen, Denmark).

[0203] The present invention also includes yet other generations, progeny, and / or offspring of the Pichia host cells of the present invention, which can be obtained, for example, by cell division.

[0204] Pharmaceutical preparations The present invention also relates to the immunoglobulin monovariable domain and / or polypeptide of the present invention, which can be obtained by the methods of the present invention as described herein.

[0205] Accordingly, the present invention also relates to pharmaceutical preparations and other compositions comprising the immunoglobulin monovariable domain and / or polypeptide of the present invention, which can be obtained by the method of the present invention. The present invention also relates to the medical use of the immunoglobulin monovariable domain and / or polypeptide of the present invention, which can be obtained by the method of the present invention.

[0206] Those skilled in the art can readily formulate pharmaceutically appropriate formulations based on general knowledge. Furthermore, express reference is made to literature specifically dealing with immunoglobulin monovariable domains and / or nanobodies, which are cited herein. Formulations for standard routes of application can be prepared, including, but are not limited to, formulations for intranasal, intraoral, intravenous, subcutaneous, intramuscular, intraperitoneal, intravaginal, rectal, topical, or inhalation application.

[0207] Based on the present invention, those skilled in the art can also readily devise appropriate treatment methods characterized by the use of a therapeutically effective amount of the immunoglobulin monovariable domain and / or polypeptide of the present invention, which can be obtained by the method of the present invention.

[0208] The present invention is further illustrated by the following embodiments, which should not be considered as further limitations. The entire contents of all documents cited throughout this application (including references, granted patents, published patent applications, and concurrently pending patent applications), in particular with respect to the doctrines referenced herein above, are expressly incorporated herein by reference.

[0209] Examples The experimental chapter describes the surprisingly low yields observed when monovalent and polyvalent immunoglobulin monovariate domains were expressed in Pichia pastris. The inventors also observed a further decrease in yield when more than one copy of the expression cassette was present in the P. pastris genome. Furthermore, a method for increasing the expression yield of the immunoglobulin monovariate domain by enhancing HAC1 splicing expression is also described.

[0210] Example 1: Identification of accessory proteins that increase nanobody expression in Pichia pastris. 1.1 Construction of the Expression Vector Nanobody A, previously described as Sequence ID No. 7 in International Publication No. 2013 / 045707, is a bivalent nanobody consisting of optimized variable domains of two sequences of a heavy chain llama antibody. The N-terminal subunit of nanobody A is a single variable domain of VHH1-type immunoglobulin, which is specific to binding to c-Met, while the C-terminal subunit binds to human serum albumin (HSA). The head and tail of these subunits are fused using a 9G / S linker. The sequence of nanobody A (Sequence ID No. 49) is shown in Table A-1. It has been previously shown that nanobody A yields very low yields (0.2 g / L or less) when fermented in P. pastris.

[0211] A DNA fragment containing the coding information for nanobody A was cloned into the multi-cloning site of a pichia expression vector (a derivative of pPIC6a, Invitrogen) containing a blastosidine® resistance gene marker, such that the nanobody sequence was downstream of the aMF signal peptide sequence and within the reading frame. To generate a pichia clone with more than one copy number of expression cassettes in the genome, a second expression cassette of nanobody A was introduced using a unique BglII site within the pichia expression vector.

[0212] The coding sequences of the auxiliary proteins shown in Table A-2 were cloned into a pichia expression vector containing a zeosin® resistance gene marker (a derivative of pPICZa, Invitrogen) using the restriction enzymes BstBI and NotI. Auxiliary proteins containing the BstBI site within their coding sequences were then cloned using the restriction enzymes AfeI and NotI. Both the nanobodies and auxiliary proteins in the pPIC6a and pPICZa vectors were under the control of the AOX1 methanol-inducible promoter.

[0213] 1.2 Transformation of nanobody code sequences, and expression and secretion of said nanobody in Pichia hosts Transformation and expression studies of wild-type Pichia X33 were performed using standard techniques and in accordance with the "User manual for pPicZalphaA, B and C" (version D, 110801, manual section number 25-0148: Invitrogen) and Methods in Molecular Biology 2007 (Humana Press Inc.). First, P. pastris strains were transformed using appropriate expression vectors containing one or two expression cassettes of nanobody A. Transformants were grown on selective medium containing blastocydin®. By characterizing many individual colonies by qPCR, clones with one copy of the expression vector integrated into the genome and clones with more than one copy of the expression cassette integrated into the genome were selected. Nanobody expression and secretion into the medium were confirmed (Figure 1).

[0214] 1.3 Transformation of auxiliary protein coding sequences, and expression and secretion of nanobodies in Pichia pastris. Once colonies expressing the appropriate nanobody were identified, their inoculum was amplified and prepared as competent cells. These cells were then transformed using an expression vector library containing the 22 accessory proteins shown in Table A-2. The transformants were grown on selective media containing various selection markers (zeosin®), thus obtaining cotransformers containing both the nanobody of interest and one or more accessory proteins from Table A-2. Expression in a shaking flask was performed in 5 mL of BMCM medium culture medium and induced by the addition of methanol as described in the Pichia protocol (see, for example, Methods in molecular biology 2007, Humana Press Inc.).

[0215] In each setting, 1128 clones were screened for improved expression and compared to their corresponding reference clones, which contained only one or more copies of a nanobody expression cassette integrated into the genome but did not contain an expression vector encoding one or more accessory proteins. In each setting, the inventors discovered two clones with significantly higher yields than their reference clones. Clones 6H1 and 4C2 had one copy of the nanobody coding sequence integrated into the genome (copy number = 1), while clones 5A6 and 9C4 had more than one copy of the nanobody coding sequence integrated into the genome (copy number > 1) (Figure 2).

[0216] 1.4 Identification of accessory proteins that have a positive effect on expression yield The identification of accessory proteins that positively affect nanobody expression yield in P. pastris was performed using genomic DNA PCR with sequence-specific PCR primers. A list of the primers used is shown in Table A-3. The identified accessory proteins are shown in Table 1.

[0217] [Table 1]

[0218] 1.5 Determining the expression yield of various clones The expression yield of nanobody / auxiliary protein(s) cotransformers was compared to the expression yield of the control (nanobody transformants without enhanced expression of one or more auxiliary proteins(s)) in expression experiments by quantifying the yield of nanobody expressed and secreted into the culture medium. Standard fed-batch fermentation conditions were used. Glycerol fed-batch culture was performed, and induction was initiated by the addition of methanol. Production was carried out on a 2 L scale in a compound medium at pH 6 and 30°C with a methanol feed rate of 4 ml / L / h.

[0219] The samples were subjected to SDS-PAGE analysis. Relative quantification of proteins was performed using concentration scan measurements of Coomassi-stained SDS-PAGE (Table 2).

[0220] [Table 2]

[0221] Clones 4C2 and 6H1 showed a significant increase in expression compared to their reference clone (a one-copy-number expression cassette integrated into the genome). This increase in expression appeared to be a result of co-expression of PDI1 present in both clones. Similarly, clones 5A6 and 9C4 showed a huge increase in yield compared to their reference clone. The co-proteins expressed in both clones 5A6 and 9C4 were Kar2p, RPP0, and the HAC1 splicing form. These co-proteins are most likely responsible for the improved expression of nanobody A. Interestingly, the expression level of clone 4C2 was significantly higher than that of clone 6H1, which appears to be a result of co-expression of Kar2p and the HAC1 splicing form, which are also present in 5A6 and 9C4, the clones showing the highest expression. All clones showing the highest yield co-expressed the HAC1 splicing form (Tables 1 and 2).

[0222] Example 2: Evaluation of nanobody A yield when the expression of individual accessory proteins is enhanced. Individual accessory proteins PDI1, Kar2p, RPP0, and HAC1 splicing forms were transformed into reference clones having more than one copy of a nanobody expression cassette in the genome, as described in Example 1.3. The transformants were grown on selective medium containing zeosin®. Cotransformers containing both nanobody A and specific accessory proteins were obtained. Expression in a shaking flask was performed in a 5 mL culture medium of BMCM medium and induced by the addition of methanol as described in the Pichia protocol (see, e.g., Methods in molecular biology 2007, Humana Press Inc.). Relative quantification of proteins was performed using concentration scan measurements on Coomassi-stained SDS-PAGE (Figure 3). All clones co-expressing one of the accessory proteins showed a significant increase in the yield of nanobody A. Here again, we observed that clones containing the HAC1 splicing form showed the greatest improvement in yield.

[0223] Example 3: Evaluation of nanobody B yield when the expression of individual accessory proteins is enhanced. 3.1 Construction of the Expression Vector Nanobody B is a trivalent nanobody consisting of three optimized variable domains of a heavy chain llama antibody sequence. The subunit within nanobody B is not a single variable domain of VHH1-type immunoglobulin and does not bind to human serum albumin (HSA). The subunit is fused head and tail using a 35 G / S linker. Nanobody B has been shown to yield very low yields (0.29 g / L or less) when fermented in P. pastris.

[0224] A DNA fragment containing the coding information for nanobody B was cloned into a multi-cloning site of a pichia expression vector containing a zeosin® resistance gene marker (a derivative of pPpT4_Alpha_S: Naatsaari et al. 2012, PLoS One 7: e39720) so that the nanobody sequence was downstream of the aMF signal peptide sequence and within the reading frame. The coding sequences for the auxiliary proteins HAC1 splicing, Kar2p, PDI1, and RPP0 were cloned into a pichia expression vector containing a blastosidine® resistance gene marker. Both the nanobody and auxiliary proteins were under the control of the AOX1 methanol-inducible promoter.

[0225] 3.2 Transformation The individual accessory proteins PDI1, Kar2p, RPP0, and HAC1 splicing forms were transformed into Pichia pastoris strain NRRL Y-11430 (ATCC number 76273). The transformants were grown on selective medium containing blastocydin®. Single clones were isolated and subsequently transformed using nanobody B.

[0226] 3.3 Determination of Expression Yield of Individual Clones Expression analysis was performed as described in Example 1.5. Relative quantification of proteins was performed using concentration scan measurements on Coomassi-stained SDS-PAGE (Table 3). Only clones co-expressing the HAC1 splicing auxiliary protein showed a significant increase in nanobody B yield, again demonstrating that enhanced expression of the HAC1 splicing auxiliary protein most effectively improves yield.

[0227] [Table 3]

[0228] Example 4: Evaluation of the yield of nanobody C showing good expression when the expression of individual accessory proteins is enhanced. Nanobody C is a bivalent nanobody consisting of two optimized variable domains of a heavy chain rama antibody sequence. The subunit within nanobody C is not a VHH1 type immunoglobulin single variable domain. The C-terminal subunit binds to human serum albumin (HSA). The subunit is fused head-tail using a 35 G / S linker. The individual accessory proteins PDI1, Kar2p, RPP0, and HAC1 splicing forms were cloned as described in Example 3 and transformed into Pichia pastris strain NRRL Y-11430. Transformants were grown on selective medium containing blastocydin™. Single clones were isolated and subsequently transformed with nanobody C. Expression analysis was performed as described in Example 1.5. Relative quantification of proteins was performed using concentration scan measurements on Coomassie-stained SDS-PAGE (Table 4).

[0229] Only clones co-expressing the HAC1 splicing type and the auxiliary protein showed a significant increase in nanobody C yield. This again indicates that enhanced expression of the HAC1 splicing type of the auxiliary protein is most effective in improving nanobody yield. This also indicates that enhanced expression of the HAC1 splicing type of the auxiliary protein can increase the yield of nanobody that exhibits good expression.

[0230] [Table 4]

[0231] Example 5: Evaluation of the expression yield of various nanobodies (nanobody D, nanobody E, nanobody F, nanobody G, and nanobody H) when the expression of the auxiliary protein HAC1 splicing type is enhanced. Nanobody D is a bivalent nanobody consisting of optimized variable domains of two sequences of a heavy chain llama antibody. The N-terminal subunit within nanobody D is a single variable domain of VHH1 immunoglobulin, specific for binding to c-Met, while the C-terminal subunit binds to human serum albumin (HSA). This subunit is fused head and tail using a 9G / S linker and contains a C-terminal Flag3-His6 epitope tag. The sequence of nanobody D (SEQ ID NO: 50) is shown in Table A-1. Nanobody E is a trivalent nanobody consisting of optimized variable domains of three sequences of a heavy chain llama antibody. The C-terminal subunit within nanobody E is a single variable domain of VHH1 immunoglobulin, while the central subunit binds to human serum albumin (HSA). This subunit is fused head and tail using a G / S linker and contains a C-terminal Flag3-His6 epitope tag. The sequence of nanobody E (SEQ ID NO: 51) is shown in Table A-1. Nanobody F is a trivalent nanobody consisting of optimized variable domains of three sequences of the heavy chain llama antibody. The C-terminal subunit within nanobody F is a single variable domain of VHH1 immunoglobulin. The subunit within nanobody F does not bind to human serum albumin (HSA). The head and tail of this subunit are fused using a 35G / S linker and contain a C-terminal Flag3-His6 epitope tag. The sequence of nanobody F (SEQ ID NO: 52) is shown in Table A-1. Nanobodies G and H are tetravalent nanobodies consisting of optimized variable domains of four sequences of the heavy chain llama antibody. The C-terminal subunits within nanobodies G and H are single variable domains of VHH1 immunoglobulin, but one of the central subunits binds to human serum albumin (HSA). The head and tail of this subunit are fused using a 35G / S linker. The sequences of nanobodies G and H (sequences 53 and 54, respectively) are shown in Table A-1. Nanobodies I are monovalent nanobodies that specifically bind to TNF. Nanobodies I consist substantially of a single sequence of optimized variable domains, further containing one alanine residue as a C-terminal extension. Nanobodies I are not a single variable domain of VHH1 immunoglobulin. The sequence of nanobodies I (sequence number 55) is shown in Table A-1.

[0232] The HAC1 splicing forms of the individual auxiliary proteins were cloned as described in 3.1 and transformed into Pichia pastoris strain NRRL Y-11430. The transformants were grown on a selection medium containing blasticidin (trademark). Single clones were isolated and subsequently transformed with nanobodies D, E, F, G, or H. Expression analysis was performed as described in Example 1.5. Relative quantification of the proteins was performed using densitometric scanning of Coomassie-stained SDS-PAGE (Table 5). Enhancement of the expression of the HAC1 splicing form of the auxiliary protein improved the yields of all nanobodies. This also demonstrates here that enhancement of the expression of the HAC1 splicing form of the auxiliary protein effectively improves the yields of nanobodies.

[0233]

Table 5

[0234]

Table 6

[0235]

Table 7

[0236]

Table 8

[0237] Table 9

Claims

1. A pichia host comprising a gene construct comprising a nucleic acid encoding a polypeptide containing at least one immunoglobulin monovariate domain and a nucleic acid encoding a HAC1 splicing type having the sequence described in SEQ ID NO: 14, Herein, a Pichia host is provided in which at least one immunoglobulin monovariate domain is selected from VHH, humanized VHH, and camelid heavy chain variable domains.

2. A pichia host capable of expressing or expressing under appropriate conditions a polypeptide comprising at least one immunoglobulin monovariate domain, wherein the expression of the HAC1 splicing type of auxiliary protein having the sequence described in SEQ ID NO: 14 is enhanced by introducing a nucleic acid encoding the HAC1 splicing type protein into the pichia host, according to claim 1.

3. The pichia host according to claim 1 or 2, wherein the expression of the auxiliary protein HAC1 splicing type having the sequence described in SEQ ID NO: 14 is enhanced in the pichia host by introducing one or more strong promoters that control the expression of nucleic acids encoding the HAC1 splicing type protein.

4. A pichia host according to any one of claims 1 to 3, wherein a polypeptide comprising at least one immunoglobulin monovariate domain and a HAC1 splicing protein are expressed from the same gene construct or from different gene constructs.

5. A pichia host according to any one of claims 1 to 4, wherein a polypeptide and / or HAC1 splicing protein comprising at least one immunoglobulin monovariate domain is expressed from the chromosome.

6. A pichia host according to any one of claims 1 to 5, wherein the copy number of the nucleic acid encoding a polypeptide containing at least one immunoglobulin monovariate domain is 1, 2 or more.

7. A pichia host according to any one of claims 1 to 6, wherein the pichia host is Pichia pastoris.

8. The Pichia host according to claim 7, wherein the Pichia pastris strain is selected from the group consisting of X33, GS115, KM71, KM71H, SMD1163, SMD1165, SMD1168, SMD1168H, NRRL-Y-11430, and GS200; or the Pichia pastris strain is selected from Pichia pastris X33 and Pichia pastris NRRLY-11430.

9. A nucleic acid encoding a polypeptide comprising at least one immunoglobulin monovariable domain and having the sequence described in SEQ ID NO: 14, Herein, a nucleic acid in which at least one immunoglobulin monovariate domain is selected from VHH, humanized VHH, and camelid heavy chain variable domains.

10. A gene construct comprising a nucleic acid encoding a polypeptide containing at least one immunoglobulin monovariable domain and a nucleic acid encoding a HAC1 splicing protein having the sequence described in SEQ ID NO: 14, Herein, a gene construct in which at least one immunoglobulin monovariate domain is selected from VHH, humanized VHH, and camelid heavy chain variable domains.

11. A pichia host according to any one of claims 1 to 8, wherein the expression of one or more additional accessory proteins is enhanced.

12. The nucleic acid according to claim 9, wherein the expression of one or more additional accessory proteins is enhanced.

13. The gene construct according to claim 10, wherein the expression of one or more additional accessory proteins is enhanced.

14. The Pichia host according to claim 11, wherein the additional co-protein is selected from PDI1, Kar2p, and RPP0.

15. The pichia host according to any one of claims 1 to 8, 11, and 14, wherein the polypeptide further comprises one or more other residues or binding units.

16. The Pichia host according to claim 15, wherein one or more other residues or binding units are linked via one or more peptide linkers.

17. A pichia host according to claim 15 or 16, wherein the one or more other binding units provide a polypeptide having an extended half-life compared to a polypeptide that does not contain the one or more binding units, A pichia host in which one or more other binding units that provide a polypeptide having an extended half-life are selected from the group consisting of binding units capable of binding to serum albumin or serum immunoglobulin.

18. The Pichia host according to claim 17, wherein the serum albumin is human serum albumin, or the serum immunoglobulin is IgG.

19. The Pichia host according to any one of claims 1 to 8, 11, and 14 to 18, wherein the polypeptide substantially consists of at least one immunoglobulin monovariate domain.

20. The Pichia host according to any one of claims 1 to 8, 11, and 14 to 19, wherein the polypeptide is a monovalent, divalent, trivalent, or tetravalent polypeptide, or a polyvalent construct.

21. A method for generating a polypeptide comprising at least one immunoglobulin monovariable domain, the method comprising the steps of expressing the polypeptide in a pichia host according to any one of claims 1 to 8, 11 and 14 to 20, and simultaneously enhancing the expression of the HAC1 splicing type auxiliary protein having the sequence described in SEQ ID NO: 14 in the pichia host, Herein, a method in which at least one immunoglobulin monovariate domain is selected from VHH, humanized VHH, and camelid heavy chain variable domains.

22. The method according to claim 21, wherein the expression of one or more additional accessory proteins is enhanced.

23. The method according to claim 22, wherein the additional co-protein is selected from PDI1, Kar2p, and RPP0.

24. The method according to any one of claims 21 to 23, wherein the polypeptide further comprises one or more other residues or binding units.

25. The method according to claim 24, wherein one or more other residues or binding units are linked via one or more peptide linkers.

26. The method according to claim 24 or 25, wherein the one or more other binding units provide a polypeptide having an extended half-life compared to a polypeptide that does not contain the one or more binding units, A method for providing a polypeptide having an extended half-life, wherein the one or more other binding units are selected from the group consisting of binding units capable of binding to serum albumin or serum immunoglobulin.

27. The method according to claim 26, wherein the serum albumin is human serum albumin, or the serum immunoglobulin is IgG.

28. The method according to any one of claims 21 to 27, wherein the polypeptide substantially consists of at least one immunoglobulin monovariable domain.

29. The method according to any one of claims 21 to 28, wherein the polypeptide is a monovalent, divalent, trivalent, or tetravalent polypeptide, or a polyvalent construct.

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