Engineering of immunoglobulin domains
10633430 · 2020-04-28
Assignee
Inventors
Cpc classification
C40B40/10
CHEMISTRY; METALLURGY
C07K2317/569
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
A61P43/00
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C40B40/10
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
Abstract
The present invention provides a single domain antibody (sdAb) scaffold comprising one or more than one non-canonical disulfide bond in the framework region (FR). The one or more than one non-canonical disulfide bond may be formed between cysteines introduced by mutations in FR2 and FR3. In the case where the sdAb scaffold is a V.sub.H, the Cys may be introduced at any one of positions 47-49 and any one of positions 67-71, based on Kabat numbering; in one example, the Cys may be introduced at positions 49 and 69, based on Kabat numbering. In the case where the sdAb scaffold is a V.sub.L, the Cys residues may be introduced at any one of positions 46-49 and any one of positions 62-66, based on Kabat numbering; in one example, the Cys residues may be introduced at positions 48 and 64, based on Kabat numbering.
Claims
1. A synthetic immunoglobulin scaffold comprising one or more than one non-canonical disulfide bond between framework region 2 (FR2) and framework region 3 (FR3), wherein the immunoglobulin scaffold is a human V.sub.L scaffold, and wherein the non-canonical disulfide bond is formed between cysteine residues introduced at position 48 in FR2 and position 64 in FR3, wherein the positions are based on Kabat numbering, wherein the human V.sub.L scaffold is at least 90% identical, including conservative amino acid mutations but disregarding the residues within complementarity determining regions (CDRs), to one or more of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.
2. The synthetic immunoglobulin scaffold of claim 1, wherein the synthetic immunoglobulin scaffold is immobilized onto a surface.
3. A polypeptide comprising the synthetic immunoglobulin scaffold of claim 1, selected from the group consisting of a single domain antibody (sdAb), scFv, Fab, F(ab).sub.2, and mature immunoglobulin.
4. The polypeptide of claim 3, wherein the mature immunoglobulin is selected from the group consisting of an IgG1, IgG2, IgG3, IgG4, IgE, and/or IgM.
5. A composition comprising the synthetic immunoglobulin scaffold of claim 1.
6. The synthetic immunoglobulin scaffold of claim 1, wherein the V.sub.L is of the kappa or lambda family.
7. The polypeptide of claim 3, wherein the synthetic immunoglobulin scaffolds are multimerized, wherein the resulting multimer comprises at least a dimer, a trimer, tetramer, pentamer, hexamer, heptamer or an octamer.
8. The polypeptide of claim 3, wherein the synthetic immunoglobulin scaffold is fused to an Fc fragment, a targeting sequence, a signal sequence, a purification tag, or any combination thereof.
9. The polypeptide of claim 3, wherein the synthetic immunoglobulin scaffold is linked to a cargo molecule.
10. A recombinant library comprising at least one synthetic immunoglobulin scaffold as defined in claim 1, wherein the synthetic immunoglobulin scaffold further comprises a multiplicity of suitable CDR sequences, wherein the multiplicity of CDR sequences provide a diversity of at least 10.sup.5, at least 10.sup.6, at least 10.sup.7, at least 10.sup.8, at least 10.sup.9 or more than 10.sup.9 clones per library.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) These and other features of the invention will now be described by way of example, with reference to the appended drawings, wherein:
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DETAILED DESCRIPTION OF THE INVENTION
(19) The present invention relates to engineering of immunoglobulin domains. More specifically, the present invention is directed to engineering of immunoglobulin domains to improve biophysical properties of the immunoglobulin. Compositions of matter and methods of making and using such compositions are provided.
(20) The present invention provides a composition comprising a polypeptide comprising a V.sub.H or V.sub.L scaffold comprising one or more than one non-canonical disulfide bond in the framework region (FR), wherein the non-canonical disulfide bond provides improved stability and non-aggregation of the polypeptide. In one embodiment the polypeptide is a V.sub.H or V.sub.L scaffold. In one embodiment, the polypeptide comprises the sdAb fragment of a V.sub.H or V.sub.L domain. The sdAb may be derived from a V.sub.H region, a V.sub.HH region or a V.sub.L region as described herein.
(21) The immunoglobulin scaffold may comprise the variable region of a heavy chain (V.sub.H). In one embodiment the V.sub.H is derived from the V.sub.H1, the V.sub.H2 or the V.sub.H3 family. In one embodiment the V.sub.H may be of the V.sub.H3 family. Alternatively, the immunoglobulin scaffold may be a variable region of a light chain (V.sub.L). In one embodiment the V.sub.L may be of the kappa or lambda family. In one embodiment the immunoglobulin scaffold is a non-human animal. In one embodiment, the non-human animal includes all vertebrates, especially vertebrates selected from avians, amphibians, reptiles, mammals, camelids, chicken, rat, mouse, rabbit, goat, sheep, cow, cat, dog, horse, or nonhuman primates. In one embodiment the immunoglobulin scaffold is human. In one embodiment the immunoglobulin scaffold is camelid. In one embodiment, the camelid species is selected from the group consisting of llama, alpaca and camel. In one embodiment, the camelid scaffold is derived from any one or more of a V.sub.H region, a V.sub.L or a V.sub.HH region.
(22) In one embodiment the immunoglobulin scaffold is humaneered (also alternatively termed humanized), i.e., a sdAb that originated from a species other than human that has had immunogenic or potentially immunogenic amino acid residues replaced with amino acids that are less immunogenic or not immunogenic in the context of a sdAb administered to a human subject. Any method known in the art for creating humaneered antibodies are contemplated in the invention, including but not limited to humaneering technology of Kalobios. Note that when a scaffold is humaneered, an immunogenic amino acid residue may be replaced by any other less immunogenic amino acid residue regardless of whether or not this constitutes a conserved amino acid change. It is understood that humaneered scaffolds of the invention retain the non-canonical Cys residues described herein, as well as any native (canonical) disulfide bonds.
(23) As used herein, the term V.sub.H or V.sub.L, also referred to herein as V.sub.H or V.sub.L domains refer to the variable region of an antibody heavy chain or an antibody light chain, respectively. In one embodiment, the V.sub.H or V.sub.L domain is in the format of a single domain antibody (sdAb). As used herein, sdAb refers to a single immunoglobulin domain that retains the immunoglobulin fold; i.e., it is a variable domain in which up to three complementarity determining regions (CDR) along with up to four framework regions (FR) form the antigen-binding site. The CDR of the V.sub.H or V.sub.L variable domain are referred to herein as CDR1, CDR2, and CDR3. The FRs of the V.sub.H or V.sub.L variable domain are referred to herein as FR1, FR2, FR3 and FR4. Various schemes exist for identification of the complementarity-determining regions, the two most common being those of Kabat and of Chothia and Lesk. Kabat et al (1991) define the complementarity-determining regions (CDR) based on sequence variability at the antigen-binding regions of the V.sub.H and/or V.sub.L domains. Chothia and Lesk (1987) define the hypervariable loops (H or L) based on the location of the structural loop regions in the V.sub.H and V.sub.L domains. As these individual schemes define CDR and hypervariable loop regions that are adjacent or overlapping, those of skill in the antibody art often utilize the terms CDR and hypervariable loop interchangeably, and they may be so used herein. The majority of the sequence variability in variable domains (V.sub.H or V.sub.L) occurs in the CDR/loops; the regions outside the CDR/loops are referred to as the framework regions (FR). The FR provide structural integrity to the variable domain and ensure retention of the immunoglobulin fold. The FR and CDR/loops are defined herein according to the Kabat numbering system (Kabat et al, 1991). In one embodiment, the numbering of the positions with the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 regions of the polypeptides described herein are as provided in
(24) Human V.sub.H or V.sub.L domains may be obtained from human Ig heavy or light chain sequences (Holliger, and Hudson, 2005; Holt, et al., 2003; Jespers et al, 2004; To et al, 2005). Similar techniques are known in the art for obtaining V.sub.H or V.sub.L domains from non-human species. Furthermore, the inventive V.sub.H and V.sub.L domains of the present invention include recombinantly produced V.sub.H or V.sub.L, as well as those V.sub.H or V.sub.L generated through further modification of such V.sub.H or V.sub.L by affinity maturation, stabilization, solubilization (e.g., camelization), or other methods of antibody engineering. Also encompassed by the present invention are homologues, derivatives, or variants that retain or improve the stability and non-aggregation characteristics of the V.sub.H or V.sub.L.
(25) The invention provides novel V.sub.H or V.sub.L domains that comprise a scaffold comprising non-canonical Cys resides. As used herein a scaffold or alternatively an immunoglobulin scaffold comprises the framework regions (FR1, FR2, FR3, and FR4) of the V.sub.H or V.sub.L, wherein the framework provides the polypeptide backbone for the various CDRs (CDR1, CDR2, CDR3). As used herein, the term scaffold ignores the CDR regions for purposes of describing the framework portion of composition of the invention.
(26) The immunoglobulin scaffold described herein may comprise a polypeptide sequence comprising at least two non-canonical Cys residues introduced into the framework regions FR2 and FR3 of an antibody variable region. In one embodiment, the polypeptide comprises a Cys residue at a position selected from residues 47 to 49 of a V.sub.H FR2 regions and a Cys residue at a position selected from residues 69 to 71 of a V.sub.H FR3 regions of a V.sub.H sdAb domain. In one embodiment, the polypeptide comprises a Cys residue at a position selected from residues 46 to 49 of a V.sub.L FR2 regions and a Cys residue at a position selected from residues 62 to 66 of a V.sub.L FR3 regions of a V.sub.L sdAb domain. As used herein, introduced or introducing means incorporating a change into the polypeptide composition or the nucleotide sequence encoding the polypeptide composition using any suitable method known to one skilled in the art, and especially including recombinant technology. In one embodiment, Cys residues are introduced into the V.sub.H and/or V.sub.L construct by replacing an existing codon in a gene, a coding sequence and/or an mRNA with a codon that is specific for a cysteine residue.
(27) In one embodiment, the inventive composition comprises an immunoglobulin scaffold selected from a V.sub.H or a V.sub.L scaffold, wherein the V.sub.H scaffold comprises at least one non-canonical disulfide bond in the FR, wherein the non-canonical disulfide bond is formed between Cys residues introduced at positions 49 and 69, based on Kabat numbering; and the V.sub.L scaffold comprises at least one non-canonical disulfide bond in the FR, wherein the non-canonical disulfide bond is formed between Cys residues introduced at positions 48 and 64, based on Kabat numbering.
(28) As used herein, an inventive V.sub.H construct or sdAb fragment comprises at least one non-canonical disulfide bridge comprising at least a Cys residue at position 49 and at least a Cys residue at position 69.
(29) As used herein, an inventive V.sub.L construct or sdAb fragment comprises at least one non-canonical disulfide bridge comprising at least a Cys residue at position 48 and at least a Cys residue at position 64.
(30) In one embodiment, the polypeptide comprises at least a Cys residue at position 49 and at least a Cys residue at position 69 of a V.sub.H domain. In one embodiment, the inventive V.sub.H domain comprises a sdAb fragment. In one embodiment, the invention provides an expression library comprising the inventive V.sub.H immunoglobulin scaffold, wherein the immunoglobulin scaffold further comprise a multiplicity of CDR sequences. In one embodiment, the invention provides a recombinant library comprising the inventive V.sub.H immunoglobulin scaffold and a multiplicity of suitable CDR sequences so as to provide libraries with diversities of at least 10.sup.5, at least 10.sup.6, at least 10.sup.7, at least 10.sup.8, at least 10.sup.9 or more than 10.sup.9 clones per library. In one embodiment, the position of the CDR regions of the inventive V.sub.H sdAb fragment selected from the group of SEQ ID NOS: 2, 4, 6 and 8 are at the positions provided in
(31) In one embodiment, the polypeptide comprises at least a Cys residue at position 48 and at least a Cys residue at position 64 of a V.sub.L domain. In one embodiment, the inventive V.sub.L domain comprises a sdAb fragment. In one embodiment, the invention provides an expression library comprising the inventive V.sub.L sdAb fragment, wherein the inventive sdAb fragments comprise a multiplicity of CDR sequences. In one embodiment, the invention provides a recombinant library comprising the inventive V.sub.L immunoglobulin scaffold and a multiplicity of suitable CDR sequences so as to provide libraries with diversities of at least 10.sup.5, at least 10.sup.6, at least 10.sup.7, at least 10.sup.8, at least 10.sup.9 or more than 10.sup.9 clones per library. In one embodiment, the position of the CDR regions of the inventive V.sub.L sdAb fragment selected from the group of SEQ ID NOS: 10, 12, 14, 16, 18, 20, 22 and 24 are at the positions provided in
(32) In one embodiment, the CDR residues of the inventive polypeptides may be any suitable sequence and may have a variable number of residues other than the number provided in
(33) The V.sub.H or V.sub.L compositions of the present invention provide scaffolds in the construction of additional libraries or antibodies. In one embodiment the framework regions (FR1, FR2, FR3, and FR4) of the V.sub.H or V.sub.L may be used to carry various CDRs, i.e., wherein the FR are as provided and wherein the sequence and number of residues of the CDR region are as provided or vary at one or more, or at all, of the CDR-specific residues. In this manner, synthetic V.sub.H or V.sub.L libraries may be constructed on single scaffolds by inserting suitable oligonucleotides comprising randomized sequences to provide randomized CDR/loops. Such libraries may be display libraries such as phage display, ribosome display, or yeast display. This approach is routine in the art and has been described in many publications (for example, but not limited to Arbabi-Ghahroudi et al., 2009a and references therein). The libraries may optionally be used for further antibody engineering or for in vitro affinity maturation (Davies and Riechmann, 1996b; Yau et al., 2005). Alternatively, an existing library based on a naturally-occurring sdAb can be used to engineer disulfide linkage(s) into every member of the library by splice overlap extension-polymerase chain reaction (SOE-PCR) (Arbabi-Ghahroudi, et al., 2010 and references therein; Ho et al., 1989) or other methods (Kunkel et al., 1987; Sidhu et al., 2000).
(34) In yet another alternative, the V.sub.H or V.sub.L may be used as a scaffold to incorporate specific CDR sequences; for example, and without wishing to be limiting in any manner, a humanized antibody may be constructed from any non-human antibody by using recombinant technology to further comprise the non-canonical disulfide bridge according to the methods provided herein. This construct may then be further modified to bind a specific target by inserting the appropriate CDR coding segments (responsible for the desired binding properties, and which may vary in sequence and in length according to guidelines cited herein and known to those skilled in the antibody arts) into the humanized antibody scaffold according to the present invention. As would be known to a person of skill in the art, these techniques are achieved through standard cloning and recombinant DNA methods using an appropriate vectors and expression in cells (mammalian or otherwise). Such methods and techniques are well-known to those of skill in the art, and are not described here.
(35) The V.sub.H or V.sub.L scaffolds of the present invention comprise one or more than one non-canonical disulfide bond in the framework regions. A disulfide bond is a covalent force that stabilizes proteins through coupling of two thiol groups between cysteine residues (Betz, 1993). By non-canonical, it is meant that the disulfide bond is not found in naturally-occurring human V.sub.H and V.sub.L, but rather is introduced using molecular engineering techniques. The naturally-occurring (or canonical) disulfide bond connecting two -sheets within a variable domain is highly conserved in V.sub.H and V.sub.L superfamilies (Amzel and Poljak, 1979; Williams and Barclay, 1988). The conserved disulfide bonds form between Cys22 and Cys92 in V.sub.Hs and Cys23 and Cys88 in V.sub.Ls, wherein the residues are numbered according to Kabat (see
(36) The Cys may be introduced into the framework regions of V.sub.H or V.sub.L domains using any suitable method known in the art. Without wishing to be limiting, the Cys may be introduced by point-mutation or insertion through recombinant DNA methods; for example and without wishing to be limiting in any manner, the Cys may be introduced using splicing by overlap extension (SOE)-PCR (Arbabi-Ghahroudi, et al., 2010 and references therein; Ho et al., 1989).
(37) The one or more than one non-canonical disulfide bond in the V.sub.H and/or V.sub.L scaffolds lends improved stability to and non-aggregation of the antibody. Non-aggregation refers to the molecule existing in a monomeric state; for example, and without wishing to be limiting in any manner, monomers give essentially one peak with size exclusion chromatography (SEC). Aggregating molecules form dimers, trimers, and higher order aggregates. Stability of an antibody includes biophysical characteristics such as thermostability, thermal and chemical refolding efficiency, and protease resistance. Other factors to be considered when evaluating the V.sub.H or V.sub.L scaffolds of the present invention include expression levels and solubility. As is known in the art, solubility refers to the number of molecules per volume (e.g., in molarity or mg/mL) that may be dissolved in a liquid prior to precipitation; in the case of sdAbs, and in the context of the present invention, the number of monomeric molecules is of particular importance.
(38) The addition of a non-canonical disulfide bond may improve one or more than one of the aforementioned biophysical characteristics. For example, and without wishing to be bound by theory, the introduction of a non-canonical disulfide bond into a V.sub.H or V.sub.L domain may improve stability (for example, higher melting temperature and protease resistance, by stabilizing the framework); and/or improve non-aggregation (by reducing inter-molecular interactions and formation of aggregates).
(39) The V.sub.H or V.sub.L scaffold into which non-canonical disulfide bonds are introduced may be of any suitable germline origin. For example, and without wishing to be limiting in any manner, the V.sub.L may be of the lambda or kappa family, for example kappa 1 or kappa 3, or its composition may be derived from various combinations of V and J segment germline sequences. In another example, and without wishing to be limiting in any manner, the V.sub.H may be of the V.sub.H1, the V.sub.H2, the V.sub.H3, the V.sub.H4, the V.sub.H5, the V.sub.H6 or the V.sub.H7 family, or its composition may be derived from various combinations of V, D and J segment germline sequences (To et al., 2005; V BASE database of the MRC Centre for Protein Engineering). In one embodiment, the V.sub.H may be of the V.sub.H3 family.
(40) In one embodiment, the V.sub.H or V.sub.L scaffolds of the present invention may include, but are not limited to scaffolds comprising the framework regions of sequences selected from the group consisting of
(41) TABLE-US-00002 (HVHAm302S;SEQIDNO:2) QVQLVESGGGLIKPGGSLRLSCAASGDTVSDESMTWVRQAPGKGLEWV AISSSGGSTYYADSVKGRFT
SRDNSKNTVYLQMNSLRAEDTAVYYCVT DNRSCQTSLCTSTTRSWGQGTMVTVSS; (HVHAm427S;SEQIDNO:4) QVQLVESGGGLIKPGGSLRLSCAASGVTLSPECMAWVRQAPGKGLEWV
AISSSGGSTYYADSVKGRFT
SRDNSKNTVYLQMNSLRAEDTAVYYCVS CEGENAFWGQGTMVTVSS; (HVHAm431S;SEQIDNO:6) QVQLVESGGGLIKPGGSLRLSCAASGYTVSSECMGWVRQAPGKGLEWV
AISSSSGSTYYADSVKGRFT
SRDNSKNIVYLQMNSLRAEDTAVYYCVR DSKNCHDKDCTRPYCSWGQGTMVTVSS; (HVHPC235S;SEQIDNO:8) QVQLVESGGGLIKPGGSLRLSCAASGFSVISESMTWVRQAPGKGLEWV
AISSSGGSTYYADSVKGRFT
SRDNSKNTVHLQMNSLRAEDTAVYYCAA KKIDGARYDYWGQGTMVTVSS (HVLP324S;SEQIDNO:10) DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLL
FAASTLQSGVPSRFS
SGSGTDFTLTISNLQPEDFATYYCQQSYSTPR TFGHGTKVTVL; (HVLP325S;SEQIDNO:12) EIVLTQSPTTLSLSPGERATLSCRASQSVGRYLAWYQQRPGQAPRLL
FDTSNRAPGVPARFS
RGSGTLFTLTISSLEPEDSAVYFCQQRSSGLT FGGGTKVTVL; (HVLP335S;SEQIDNO:14) EIVMTQSPATLSLSPGERATLSCRASQSVSSSSLAWYQQKPGQAPRLL
YGTSNRATGIPDRFS
SGSGTHFTLTINRLEPGDFAVYYCQQYGSSPRT FGQGTKVEIK; (HVLP342S;SEQIDNO:16) DIQMTQSPSSLSASVGDRVTITCRASQDIRTDLDWFQQRPGRAPHRL
YGASSLQGGVPSRFS
SGSGTEFTLTISGLQPEDFATYYCLQHHTYPR TFGLGTKVTVL; (HVLP351S;SEQIDNO:18) EIVMTQSPVTLSLSPGERATLSCRASQSVGTSLAWYQQKPGQAPRLL
YDASNRATGISARFS
SGSGTDFTLTISSLEPEDFAVYYCQQRYNWPR TFGGGTKVTVL; (HVLP364S;SEQIDNO:20) ETTLTQSPATLSVSPGERATFSCRASQSVSNNLAWYQQKPGQAPRLL
YGASSRTTGIPDRFS
SGSGTDFTLTISRLEPEDFAVYYCQQYDTSPR TFGQGTKVEIK; (HVLP389S;SEQIDNO:22) QSVVTQPPSVSAAPGQRVTISCSGSSYNIGENSVSWYQQLPGTAPKLL
YGNDKRPSGIPDRFS
SKSGTSATLGITGLQTGDEADYYCGTWDSNLRA SVFGGGTKVTVL; (HVLP3103S;SEQIDNO:24) ETTLTQSPGTLSLSPGERATLSCRASQSVRNNLAWYQQRPGQAPRLL
YGASTRATGIPARFS
SGSGTDFTLTISSLQVEDVAVYYCQQYYTTPK TFGQGTKVEIK, (SEQIDNO:70 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV
AISGSGGSTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAEDTAVYYCAK DEPRSVSGLRGVVDSWGRGTLVTVSS (SEQIDNO:71) QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV
AISGSGGSTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAEDTAVYYCGT DMEVWGKGTTVTVSS (SEQIDNO:72) QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV
AISGSGGSTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAEDTAVYYCAK DGKGGSSGYDHPDYWGQGTLVTVSS (SEQIDNO:73) QLQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV
AISGSGGSTYYADSVKGRFT
SRDNSKNSLYLQMNSLGAEDTAVYYCAR SWSGSSYGGDLDSWGQGTLVTVSS (SEQIDNO:74) QVQLVESGGGLIKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWV
AISSSGGSTYYADSVKGRFT
SRDNSKNTVYLQMNSLRAEDTAVYYCVR EEYRCSGTSCPGAFDIWGQGTMVTVSS (SEQIDNO:75) EVQLVESGGTLVQPGGSLRLSCAASGFTFINYAMSWVRQAPDKGLDWV
TISNNGGATYYADSVKGRFT
SRDNSNNTLYLQMNSLRPDDTAVYYCAK GPINTGRYGDWGQGTLVTVSS (SEQIDNO:76) QVQLVQSGGGLVQPGRSLRLSCAASGFAFSSYAMSWVRQAPGKGLEWV
AISGGGDHTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAEDTAVYYCAK EGMVRGVSSAPFDYWGQGTLVTVSS (SEQIDNO:77) EVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWV
GISGSGASTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAGDTALYYCAR QSITGPTGAFDVWGQGTMVTVSS (SEQIDNO:78) QLQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWFRQAPGKGLEWV
FIRSKAYGGTTEYAASVKGRFT
SRDDSKSIAYLQMNSLRAEDTAMYYC ARRAKDGYNSPEDYWGQGTLVTVSS (SEQIDNO:79) QVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMTWVRQAPGKGLEWV
RIKTKTDGGTTDYAAPVKGRFT
SRDDSKNTLYLQMNSLKTEDTAVYYC TTDRDHSSGSWGQGTLVTVSS (SEQIDNO:80) DVQLVQSGGGLVKPGGSLRLSCTASGFPFSNAWMSWVRQAPGKGLEWV
RITSKTDGGITDYVAPVKGRFT
SRDDSKNTLYLQMNSLKTEDTAVYYC TTDQANAFDIWGQGTMVTVSS (SEQIDNO:81) QMQLVQSGGGVVQPGGSLRLSCAASGFTVSSSRMSWFRQAPGMGLEWV
VIYSGGSTYYADSVRGRFS
SRDNSKNTLYLQMNSLRAEDTALYYCARE REGAVTREDWGQGTLVTVSS (SEQIDNO:82) QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWV
FIYSGGSTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAEDTAVYYCARE SRVGGGAFDIWGQGTMVTVSS and (SEQIDNO:83) QVQLVQSGGGVVQPGRSLRLSCAASGFIVDGYAMHWVRQAPGQGLEWV
VTNNGGSTSYADSVKGRFT
SRDNSKNTVYLQMNSLRAEDTAVYYCARQ SITGPTGAFDIWGQGTMVTVSS
or sequences substantially identical thereto, or fragments thereof, with the proviso that the substantially identical sequence, or fragments thereof, retains both the canonical and non-canonical disulfide bonds and wherein the sequence in the regions of the CDRs may be any suitable sequence and may have a suitable but variable number of residues in each of CDR1, CDR2 and CDR3. In the case of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24, the residues that comprise the CDRs are as provided in
(42) A substantially identical sequence may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered substantially identical polypeptides. Conservative amino acid mutation may include addition, deletion, or substitution of an amino acid; a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity).
(43) In a non-limiting example, a conservative mutation may be an amino acid substitution. Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term basic amino acid it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term neutral amino acid (also polar amino acid), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term hydrophobic amino acid (also non-polar amino acid) is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). Acidic amino acid refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).
(44) Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics, BLAST-P, Blast-N, or FASTA-N, or any other appropriate software that is known in the art. In one embodiment, calculation of percent identity is limited to comparison of the framework regions and disregards the residues within the CDRs. In one embodiment, calculation of percent identity compares all residues in a polypeptide, including both those of the framework regions and the CDRs.
(45) As noted throughout the text, the inventive scaffolds are not limited to the residues listed in the CDR regions, which are as delimited in
(46) The substantially identical sequences of the present invention may be at least 90% identical; in another example, the substantially identical sequences may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any percentage therebetween) identical at the amino acid level to sequences described herein. In a further embodiment, a substantially identical sequence may contain one, two, three or four amino acid differences in the framework region when aligned with an inventive composition provided herein. Importantly, the substantially identical sequences retain the stability and biophysical properties of the reference sequence. In a non-limiting embodiment, the difference in sequence identity may be due to conservative amino acid mutation(s). In one embodiment, the substantially identical sequences consist of the residues comprising FR1, FR2, FR3 and FR4 and does not consider the residues comprising any one or more of CDR1, CDR2 and CDR3.
(47) The V.sub.H or V.sub.L scaffolds may be included as part of larger antibody protein or fragments such as one or more of a sdAb, scFv, Fab, F(ab).sub.2 and/or mature immunoglobulin, including but not limited to an IgG, IgE, and/or IgM, for the purpose of increasing their biophysical properties such as non-aggregation, stability, expression level, and solubility. As used herein, a mature immunoglobulin comprises a light chain, said light chain comprising a variable and a constant region, and a heavy chain, said heavy chain comprising a variable and a constant region. In one embodiment, the mature immunoglobulin is selected from the group consisting of an IgG1, IgG2, IgG3, IgG4, IgE, and/or IgM. In one embodiment the introduced sequence of this larger antibody construct is human or is humaneered. The above approach can also be applied to V.sub.H and V.sub.L derivatives such as fusion constructs made by fusions of V.sub.Hs and V.sub.Ls to other polypeptides such as toxins, V.sub.Hs and/or V.sub.Ls with the same or different specificity, Fc domains, etc. for the purpose of increasing their biophysical properties such as solubility, non-aggregation, expression level and stability.
(48) The V.sub.H or V.sub.L scaffolds of the present invention may also be fused to additional sequences to aid in expression, detection or purification of a recombinant antibody or fragment thereof. Any such sequences or tags known to those of skill in the art may be used. For example, and without wishing to be limiting, the antibody or fragment thereof may comprise a targeting or signal sequence (for example, but not limited to ompA), a detection tag (for example, but not limited to c-Myc), a purification tag (for example, but not limited to a His.sub.5 or His.sub.6), an Fc fragment, or a combination thereof. In another example, the additional sequence may be a biotin recognition site such as that described by Cronan et al in WO 95/04069 or Voges et al in WO/2004/076670. As is also known to those of skill in the art, linker sequences may be used in conjunction with the additional sequences or tags.
(49) The V.sub.H or V.sub.L scaffolds of the present invention may also be in a multivalent display. Multimerization may be achieved by any suitable method known in the art. For example, and without wishing to be limiting in any manner, multimerization may be achieved using self-assembly molecules as described in Zhang et al (2004a; 2004b) and WO2003/046560. The described method produces pentabodies by expressing a fusion protein comprising the V.sub.H or V.sub.L scaffold of the present invention and the pentamerization domain of the B-subunit of an AB.sub.5 toxin family (Merritt and Hol, 1995); the pentamerization domain assembles into a pentamer, through which a multivalent display of the antibody or fragment thereof is formed. Additionally, the pentamerization domain may be linked to the V.sub.H or V.sub.L scaffold using a linker; such a linker should be of sufficient length and appropriate composition to provide flexible attachment of the two molecules, but should not hamper the biophysical properties of the V.sub.H or V.sub.L scaffold.
(50) Other forms of multivalent display are also encompassed by the present invention. For example, and without wishing to be limiting, the V.sub.H or V.sub.L scaffold may be presented as at least a dimer, a trimer, tetramer, pentamer, hexamer, heptamer, octamer, or any other suitable oligomer. This may be achieved by methods known in the art, for example direct linking connection (Nielson et al, 2000), c-jun/Fos interaction (de Kruif and Logtenberg, 1996), Knob into holes interaction (Ridgway et al, 1996) or sequential cloning of repeating units of the coding region of the inventive clone and suitable short polypeptide linkers known in the art.
(51) Another method known in the art for multimerization is to dimerize the V.sub.H or V.sub.L composition using an Fc domain. When applied in vivo, monomeric sdAb are cleared quickly from the circulation (Bell et al., 2010). To solve this problem and to give V.sub.H or V.sub.L the ability to induce immune response after antigen binding, V.sub.H or V.sub.L scaffolds may be fused to an antibody constant region fragment (Fc) to generate chimeric heavy chain antibodies (Bell et al., 2010). In this approach, the Fc gene in inserted into a vector along with the V.sub.H or V.sub.L gene to generate a sdAb-Fc fusion protein (Bell et al, 2010; Iqbal et al, 2010); the fusion protein is recombinantly expressed then purified. Such antibodies are easy to engineer and to produce (Zhang et al., 2009), can greatly extend the serum half life of V.sub.H or V.sub.L, and may be excellent tumor imaging reagents (Bell et al., 2010). In one embodiment, the Fc portion is human.
(52) The present invention also encompasses nucleic acid sequences encoding the molecules as described herein. In one embodiment, the nucleic acid comprises a sequence encoding an immunoglobulin scaffold selected from a V.sub.H or a V.sub.L scaffold, wherein the V.sub.H scaffold comprises at least one non-canonical disulfide bond in the FR formed between Cys residues introduced at positions 49 and 69, based on Kabat numbering; and the V.sub.L scaffold comprises at least one non-canonical disulfide bond in the FR formed between Cys residues introduced at positions 48 and 64, based on Kabat numbering. In one embodiment, the nucleic acid encodes any one or more of the sequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 70-83. In one embodiment, the nucleic acid sequence comprises any codon of the degenerate code that encodes the appropriate amino acid residue. In one embodiment, coding region of the nucleic acid sequence is codon optimized. In one embodiment, the nucleic acid is in an expression vector. In one embodiment, the expression vector is in a host cell or organism capable of expressing said nucleic acid.
(53) As is well known to those of skill in the art, it is possible to improve the expression of a nucleic acid sequence in a host cell or organism by replacing the nucleic acids coding for a particular amino acid (i.e. a codon) with another codon which is better expressed in the host organism. One reason that this effect arises is due to the fact that different organisms show preferences for different codons. In particular, bacterial organisms and yeast organisms prefer different codons from plants and animals. The process of altering the sequence of a nucleic acid to achieve better expression based on codon preference is called codon optimization. Statistical methods have been generated to analyze codon usage bias in various organisms and many computer algorithms have been developed to implement these statistical analyses in the design of codon optimized gene sequences (Lithwick and Margalit, 2003). In one embodiment, the nucleic acid sequence may be codon-optimized for expression in mammalian cells. In one embodiment, the nucleic acid sequence may be codon-optimized for expression in various micro-organisms. In one embodiment, the nucleic acid sequence is codon-optimized for expression in E. coli. In one embodiment, the nucleic acid sequence is codon-optimized for expression in yeast. In one embodiment, the nucleic acid sequence is codon-optimized for expression in yeast and for surface display of antibodies, i.e., shuttle between antibody phage display and antibody yeast display. The present invention also encompasses vectors comprising the nucleic acids as just described. Furthermore, the invention encompasses cells comprising the nucleic acid and/or vector as described.
(54) The present invention further encompasses the V.sub.H or V.sub.L scaffold immobilized onto a surface using various methodologies; for example, and without wishing to be limiting, the V.sub.H or V.sub.L scaffold may be linked or coupled to the surface via His-tag coupling, biotin binding, covalent binding, adsorption, and the like. The solid surface may be any suitable surface, for example, but not limited to the well surface of a microtiter plate, channels of surface plasmon resonance (SPR) sensorchips, membranes, beads (such as magnetic-based or sepharose-based beads or other chromatography resin), glass, a film, or any other useful surface.
(55) The present invention further provides an inventive V.sub.H or V.sub.L linked to a cargo molecule; the antibody or fragment thereof may deliver the cargo molecule to a desired site. The cargo molecule may be any type of molecule that may be use to diagnose, treat or detect a biomarker. In one embodiment, the cargo marker is a molecule that may reduce and/or inhibit the growth of targeted cells. In one embodiment, the targetted cells are cells associated with a proliferative disease. In one embodiment, the proliferative disease is a cancer, including various types of tumours, wherein the targeted cells express a marker recognized by the V.sub.H or V.sub.L domain portion of the invention. Thus, in one embodiment the cargo molecule is a therapeutic or diagnostic agent. In one embodiment the cargo molecule is linked to a therapeutic or diagnostic agent.
(56) For example, and without wishing to be limiting in any manner, the therapeutic agent may be a radioisotope, which may be used for radioimmunotherapy; a toxin, such as an immunotoxin; a cytokine, such as an immunocytokine; a cytotoxin; an apoptosis inducer; an enzyme; or any other suitable therapeutic molecule known in the art. In the alternative, a diagnostic agent may include, but is by no means limited to a radioisotope, a paramagnetic label, a fluorophore, an affinity label (for example biotin, avidin, etc), fused to a detectable protein-based molecule, or any other suitable agent that may be detected by imaging methods. In a specific, non-limiting example, the antibody or fragment thereof may be linked to a fluorescent agent such as FITC or may genetically be fused to the Enhanced Green Fluorescent Protein (EGFP).
(57) The antibody or fragment thereof may be linked to the cargo molecule using any method known in the art (recombinant technology, chemical conjugation, etc.).
(58) The present invention also provides a method of improving stability of single domain antibodies, comprising introducing one or more than one non-canonical disulfide bond in the framework region. In one embodiment, the method comprises introducing at least two non-canonical Cys residues into the framework regions of the immunoglobulin of interest. In one embodiment, the two Cys residues replace residues in the FR2 and FR3 of an antibody variable region. In one embodiment, the method comprises introducing a Cys residue at a position selected from residues 47 to 49 of a V.sub.H FR2 regions and a Cys residue at a position selected from residues 69 to 71 of a V.sub.H FR3 regions of a V.sub.H sdAb domain. In one embodiment, the method comprises introducing a Cys residue at a position selected from residues 46 to 49 of a V.sub.L FR2 regions and a Cys residue at a position selected from residues 62 to 66 of a V.sub.L FR3 regions of a V.sub.L sdAb domain.
(59) In one embodiment, the method comprises introducing at least one non-canonical Cys residue at position 49 and at least one non-canonical Cys residue at position 69 of a V.sub.H domain. In one embodiment, the method comprises introducing at least one non-canonical Cys residue at position 48 and at least one non-canonical Cys residue at position 64 of a V.sub.L domain.
(60) In one embodiment, the method comprises the creation of an expression library comprising the inventive V.sub.H sdAb fragment comprising a disulfide bridge between non-canonical Cys residues at positions 49 and 69, wherein the inventive sdAb fragments comprise a multiplicity of CDR sequences. In one embodiment, the method comprises the creation of an expression library comprising the inventive V.sub.L sdAb fragment comprising a disulfide bridge between non-canonical Cys residues at positions 48 and 64, wherein the inventive sdAb fragments comprise a multiplicity of CDR sequences. Any suitable method known in the art for antibody library creation replacement of CDR sequences may be used. In one embodiment, the position of the CDR regions are as defined by Kabat. In one embodiment, the position of the CDR regions are as defined by Chothia and Lesk. In one embodiment, the expression library is codon optimized for expression in mammalian cells. In one embodiment, the expression library is codon optimized for expression in yeast. In one embodiment, the expression library is codon optimized for expression in E. coli.
(61) In one embodiment, the mutations can be introduced by SOE-PCR approach (Arbabi-Ghahroudi et al., 2010 and reference therein; Ho et al., 1989) or by other method (Kunkel et al., 1987; Sidhu et al., 2000, Hussack et al., In press (c)). Alternatively, the genes for the mutants can be obtained commercially, either on their own or within the target cloning and expression vector (Kim et al., In press).
(62) The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.
Example 1: Cloning, Expression and Purification of Mutant VHs and VLs
(63) Aggregating and non-aggregating V.sub.H and V.sub.L domains as well as their counterpart double cysteine mutants were cloned, expressed and purified. V.sub.H and V.sub.L utilized, as well as their corresponding Cys mutants, are listed in Table 1 and shown in
(64) TABLE-US-00003 TABLE 1 V.sub.H and V.sub.L and mutants constructed. V.sub.H* V.sub.H Cys mutant HVHAm302 (SEQ ID NO: 1) HVHAm302S (SEQ ID NO: 2) HVHAm427 (SEQ ID NO: 3) HVHAm427S (SEQ ID NO: 4) HVHAm431 (SEQ ID NO: 5) HVHAm431S (SEQ ID NO: 6) HVHPC235 (SEQ ID NO: 7) HVHPC235S (SEQ ID NO: 8) HVHP44 HVHP44S (SEQ ID NO: 70) HVHB82 HVHB82S (SEQ ID NO: 71) HVHP421 HVHP421S (SEQ ID NO: 72) HVHP419 HVHP419S (SEQ ID NO: 73) HVHP430 HVHP430S (SEQ ID NO: 74) HVHP429 HVHP429S (SEQ ID NO: 75) HVHM41 HVHM41S (SEQ ID NO: 76) HVHM81 HVHM81S (SEQ ID NO: 77) HVHP428 HVHP428S (SEQ ID NO: 78) HVHP420 HVHP420S (SEQ ID NO: 79) HVHP414 HVHP414S (SEQ ID NO: 80) HVHP423 HVHP423S (SEQ ID NO: 81) HVHP413 HVHP413S (SEQ ID NO: 82) HVHP426 HVHP426S (SEQ ID NO: 83) V.sub.L* V.sub.L Cys mutant HVLP324 (SEQ ID NO: 9) HVLP324S (SEQ ID NO: 10) HVLP325 (SEQ ID NO: 11) HVLP325S (SEQ ID NO: 12) HVLP335 (SEQ ID NO: 13) HVLP335S (SEQ ID NO: 14) HVLP342 (SEQ ID NO: 15) HVLP342S (SEQ ID NO: 16) HVLP351 (SEQ ID NO: 17) HVLP351S (SEQ ID NO: 18) HVLP364 (SEQ ID NO: 19) HVLP364S (SEQ ID NO: 20) HVLP389 (SEQ ID NO: 21) HVLP389S (SEQ ID NO: 22) HVLP3103 (SEQ ID NO: 23) HVLP3103S (SEQ ID NO: 24) *Referred to herein as wild-type. If no SEQ ID NO is provided, then the wild type sequence is provided in PCT publication WO2006/099747, especially as shown in FIG. 2 of the publication.
(65) Wild-type V.sub.HS, HVHAm302, HVHAm427, HVHAm431, and HVHPC235, were prepared as described in Arbabi-Ghahroudi et al. (2009b). 14 additional wild type V.sub.HS, HVHP44, HVHB82, HVHP421, HVHP419, HVHP430, HVHP429, HVHM41, HVHM81, HVHP428, HVHP420, HVHP414, HVHP423, HVHP413, and HVHP426 and wild type V.sub.Ls were prepared as described in WO 2006/099747 by Tanha. Plasmids containing genes for these wild-type domains (excluding the 14 aforementioned V.sub.Hs; see below) were used as templates to construct corresponding mutant versions by SOE-PCR.
(66) Inventive constructs, also referred to herein as Cys mutants, of V.sub.Hs and V.sub.Ls (HVHAm302, HVHAm427, HVHAm431, and HVHPC235) were created with splicing by overlap extension (SOE)-PCR (Ho et al., 1989; Kim et al., Submitted Arbabi-Ghahroudi et al, 2010) to introduce a pair of Cys at the positions of 49 and 69 (V.sub.Hs) or positions 48 and 64 (V.sub.Ls) as defined by Kabat numbering (Kabat et al., 1991) and as provided in
(67) TABLE-US-00004 M13RPa (SEQIDNO:37) (5-TCACACAGGAAACAGCTATGAC-3) KT131 (SEQIDNO:38) (5-ACCACTACTACTAATAGCGCAGACCCACTCCAGCCCCTTC-3) (for302S-1sub-fragment) and M13FP (SEQIDNO:39) (5-CGCCAGGGTTTTCCCAGTCACGAC-3) KT129 (SEQIDNO:40) (5-GCAGACTCCGTGAAGGGCCGATTCACCTGCTCCAGAGACAAT TCCAAGAAC-3) (for302S-2sub-fragment),
(68) Secondly, using 302S-2 as a template, fragment 302S-2-2 is made by PCR using the following pair of PCR primers: M13FP and
(69) TABLE-US-00005 KT130 (SEQIDNO:41) (5-TGCGCTATTAGTAGTAGTGGTGGTAGCACATACTACGCAGACTCC GTGAAGGGCCG-3).
(70) This step added the overlapping region to 302S-2. Subsequently, sub-fragments 302S-1 and 302S-2-2 were assembled to create full-length V.sub.H fragment with Cys mutations by SOE-PCR using primer pair M13RP a or b/M13FP. Other V.sub.H mutants were generated from their corresponding wild-type V.sub.H plasmid using the same procedures and primers; V.sub.L mutants were generated using the same SOE-PCR procedure as that for the V.sub.H mutants, except different, V.sub.L gene-specific PCR primers and corresponding wild-type V.sub.L plasmid templates were used. The Cys mutant versions of the 14 non-aggregating V.sub.Hs, HVHP44, HVHB82, HVHP421, HVHP419, HVHP430, HVHP429, HVHM41, HVHM81, HVHP428, HVHP420, HVHP414, HVHP423, HVHP413, and HVHP426 were synthesized by DNA2.0 (Menlo Park, Calif., USA). They are referred to as HVHP44S, HVHB82S, HVHP421S, HVHP419S, HVHP430S, HVHP429S, HVHM41S, HVHM81S, HVHP428S, HVHP420S, HVHP414S, HVHP423S, HVHP413S, and HVHP426S, respectively. For these 14 constructs, the framework and CDR regions are otherwise the same as those of the corresponding wild-type sequences, which are as provided in PCT publication WO2006/099747, especially as shown in FIG. 2 of the publication.
(71) The full-length Cys mutants were then cloned into an expression vector, expressed, purified, and their concentrations determined as described elsewhere (Sambrook et al., 1989; To, et al., 2005; Arbabi-Ghahroudi et al 2009a; 2009b; 2010).
(72) The additional disulfide linkage had no adverse effect on the expression yield of Cys mutant V.sub.Hs and V.sub.Ls; wild-type and mutant sdAbs had comparable yield in milligram quantities. In fact, in some expression trials, HVHAm302S and HVHAm431S had significantly higher expression yields than their wild-type counterparts (see IMAC elution profiles in
(73) On non-reducing SDS-PAGE gels, the mutant V.sub.Hs and V.sub.Ls migrated more slowly than their wild-type counterparts (
Example 2: Disulfide Linkage Mapping of Cys Mutants
(74) Because the disulfide linkage introduced into the V.sub.H and V.sub.L Cys mutants is not natural to the antibody, the presence of disulfide linkage at the mutated position was verified prior to further characterization of the V.sub.H and V.sub.L mutants.
(75) Based on the knowledge of the two Cys residues (Cys22 and Cys92) forming a native disulfide linkage in the wild-type V.sub.Hs (Amzel and Poljak, 1979; Williams & Barclay, 1988) and the mutated residues of the mutants, the location of disulfide linkages could be predicted. Since there were some trypsin cleavage sites present between the predicted disulfide bonds in the V.sub.Hs, it was possible to trypsinize the Cys mutants and utilize mass spectrometry to verify the presence of the engineered disulfide linkage.
(76) Disulfide linkage determination for V.sub.Hs and V.sub.Ls was carried out as described elsewhere (Hussack et al., In press (b); Kim and Tanha, In press; Wu et al., 2009). Disulfide linkage determination for HVHAm302 and HVHAm302S were carried out exactly as described in Kim and Tanha (In press). Briefly, V.sub.H Cys mutants were concentrated in 0.1 M Tris-HCl, pH 8.5, using Ultrafree-0.5 centrifugal filter device with biomax-5 membrane (MWCO 5000; Millipore, Nepean, ON, Canada) and subjected to trypsin digestion (Roche Diagnostics Canada, Laval, QC, Canada) at concentration of 0.5 mg/mL in 0.1 M Tris-HCl, pH 8.5, and analyzed by SDS-PAGE for the success of trypsin digestion and subsequently subjected to a peptide mass spectrometry analysis (
(77) Results showed that the disulfide-linked peptides produced by tryptic digestion of V.sub.Hs could be identified by the mass spectrometry method employed (
(78) TABLE-US-00006 TABLE2 DisulfidelinkagedeterminationofV.sub.HSbymassspectrometry.Majortryptic peptidescontainingdisulfidelinkagesareshown,withconnectedcysteines depictedinboldandunderlined;theremainingsequencesintheVHarelostby trypsinization.Spaceswithinpeptidedoubletsdenotesequencediscontinuity. V.sub.HS Trypticpeptides MW.sub.for MW.sub.exp MW SEQIDNO HVHAm302 SCQTSLCTSTTR 1284.56 1284.57 0.01 25 LSCAASGDTVSDESMTWVR 3630.55 3630.52 0.02 26 AEDTAVYYCVTDNR 27 HVHAm302S SCQTSLCTSTTR 1284.56 1284.57 0.01 28 GLEWVCAISSSGGSTYYADSVKFTCSR 2889.28.sup.a 2889.30.sup.a 0.02.sup.a 29 30 LSCAASGDTVSDESMTWVR 3630.54 3630.52 0.02 31 AEDTAVYYCVTDNR 32 HVHPC235S GLEWVCAISSSGGSTYYADSVKFTCSR 2889.30.sup.a 2889.23.sup.a 0.07.sup.a 33 34 LSCAASGFSVISESMTWVR 3331.52 3331.43 0.09 35 AEDTAVYYCAAK 36 HVHP426S QAPGQGVEWVCVTNNGGSTSADSVK 3277.47.sup.a 3277.32.sup.a 0.04.sup.a 84 FTCSR 85 .sup.aThe very close match between MW.sub.for and MW.sub.exp indicates the presence of the Cys49Cys69 disulfide linkage. MW.sub.for: formula (expected) molecular weight; MW.sub.exp: molecular weight determined experimentally by MS; MW is calculated: (MW.sub.forMW.sub.exp). MW.sub.for, MW.sub.exp, and MW are given in Daltons (Da).
(79) In case of HVHAm302S, a prominent ion at m/z 964.04 (3+) was sequenced as a disulfide-linked peptide GLEWVCAISSSGGSTYYADSVK (P1) (SEQ ID NO:29) and FTCSR (P2) (SEQ ID NO:30) (
(80) As another representative, one of the 14 mutant V.sub.Hs (HVHP426S) was also chosen for the determination of the Cys 49/Cys 69 disulfide linkage formation. As in the case of other V.sub.Hs, the MS results showed that HVHP426S had formed a disulfide linkage between Cys 49 and Cys 69 (Table 2).
(81) A similar mass spectrometry analysis on the mutant V.sub.Ls confirmed the presence of the engineered disulfide linkage between positions Cys48 and Cys64 in all V.sub.Ls (Table 3).
(82) TABLE-US-00007 TABLE3 DisulfidelinkagedeterminationofV.sub.LSbymassspectrometry(MS).Majortryptic peptidescontainingdisulfidelinkagesareshown,withconnectedcysteinesdepictedbylike underlining(anditalics)andbolded;IncaseofHVLP324S,HVLP342S,HVLP351S,and HVLP3103S,anotherpairofconnectedcysteinesisitalicized;theremainingsequencesinthe V.sub.Larelostbytrypsinization.Spacesbetweenpeptidefragmentsdenotesequencediscontinuity. V.sub.LS Trypticpeptides MW.sub.for MW.sub.exp MW.sup.a SEQIDNO HVLP324S VTIT RLLCFASTLQSGVPSR 6249.91 6249.96 0.05 42,43,44 FSCSGSGTDFTLTISNQPEDFATYY
QQSYSTPR HVLP325S ATLSCRGSGTLFTLTISSLEPEDSAVYFCQQR 3495.66 3495.40 0.26 45,46 LLCFDTSNRFSCR 1576.71 1576.68 0.03 47,48 HVLP335S LLCYGTSNRFSCSGSGTHFTLTIVR 2750.29 2750.32 0.03 49,50 ATLSCRLEPGDFAVYYCQQYGSSPR 2826.27 2820.20 0.07 51,52 HVLP342S VTIT
RLCYGASSLQGGVPSR 6136.85 6136.80 0.05 53,54,55 FSCSGSGTEFTLTISGLQPEDFATYY
LQHHTYPR HVLP351S ATLS
RLLCYDASNR 5049.26 5049.00 0.26 56,57,58 FSCSGSGTDFTLTISSLEPEDFAVYY
QQR HVLP364S LLCYGASSRTRFSCSGSGTDFTLTISR 2644.22 2644.32 0.10 59,60 ATFSCRLEPEDFAVYYCQQYDTSPR 3004.30 3004.35 0.05 61,62 HVLP389S LLCYGNDKFSCSK 1492.67 1492.59 0.08 63,64 VTISCSGSSYNIGENSVSWYQQLPGTAPK 6221.84 6222.12 0.28 65 SGTSATLGITGLQTGDEADYYCGTWDSNLR 66 HVLP3103S ATLS
RLLCYGASTR 5528.56 5528.52 0.04 67,68,69 FSCSGSGTDFTLTISSLQVEDVAVYY
QQYYTTPK .sup.aThe very close match between MW.sub.for and MW.sub.exp indicates the presence of the Cys48Cys64 disulfide linkage. MW.sub.for: formula (expected) molecular weight; MW.sub.exp: molecular weight determined experimentally by MS; MW is calculated: (MW.sub.forMW.sub.exp). MW.sub.for, MW.sub.exp, and MW are given in Daltons (Da).
(83) Crystal structure analysis has confirmed the presence of the disulfide bridge between the non-canonical Cys-Cys residues in addition to the disulfide bridge between the canonical Cys-Cys residues in all inventive V.sub.H and V.sub.L constructs tested.
Example 3: Analytical Size Exclusion Chromatography
(84) Size exclusion chromatography (or gel filtration chromatography) separates proteins by molecular size and hydrodynamic volume (Porath and Flodin, 1959). Therefore, this method is useful in assessing protein aggregation status in solution. Size exclusion chromatography employing Superdex 75 is used to assess the aggregation state of V.sub.H (or V.sub.L) domains. Non-aggregating V.sub.Hs (or V.sub.L) should yield chromatograms with a single, symmetrical peak with elution volumes expected for a monomeric V.sub.Hs (or V.sub.Ls). In contrast, the chromatogram profiles of aggregating V.sub.Hs, in addition to the monomeric peaks, consist of additional peaks, e.g., large aggregates, dimeric aggregates, which elute earlier. Percent monomer can be calculated by area integration of the peaks and used as a quantitative measure of V.sub.H (or V.sub.L) aggregation tendency (the higher the % monomer of a V.sub.H (or V.sub.L), the lower its aggregation tendency and vice versa the higher the % aggregates of a V.sub.H (or V.sub.L) the higher its aggregation tendency).
(85) Size exclusion chromatography of V.sub.Hs and V.sub.Ls, as well as their corresponding Cys mutants, was carried out as previously described (Sambrook et al., 1989; To, et al., 2005; Arbabi-Ghahroudi et al., 2009a; Arbabi-Ghahroudi et al., 2009b; Arbabi-Ghahroudi et al., 2010; Kim and Tanha, In press). Briefly, a Superdex 75 size exclusion column was washed with 50 mL of filtered and degassed ddH.sub.2O and subsequently equilibrated with 50 mL of filtered and degassed PBS at a pump speed of 0.5 mL/min. Samples were filtered through 0.22 m disposable filter unit and subsequently submitted to size exclusion chromatography on AKTA FPLC using the Superdex column with PBS buffer at a flow rate of 0.5 mL/min, as per manufacturer's instructions. Eluates corresponding to peaks were collected using an AKTA fraction collector with 0.5 mL of fraction volume per tube. Following size exclusion chromatography, A.sub.280 versus elution volume was plotted using graphing software GraphPad Prism (version 4.02 for Windows; GraphPad Software, San Diego, Calif.) (
(86) Absorbance values (A.sub.280) on size exclusion chromatograms (SEC) were normalized and plotted against elution volumes. Absorbance normalization was performed according to the following formula:
% A.sub.280=(A.sub.280NA.sub.280B)/(A.sub.280MA.sub.280B).sub.100
Where % A.sub.280 is the normalized absorbance, A.sub.280N is the absorbance at any elution volume, A.sub.280M is the maximum absorbance and A.sub.280B is the baseline absorbance.
(87) Results (
(88) Wild-type V.sub.Ls were essentially free of aggregates and gave symmetric single peaks. V.sub.L Cys mutants were also monomeric with the exception of HVLP364S, which showed slight aggregation (11% dimeric aggregate). Thus, in general, the engineered disulfide linkage did not compromise the non-aggregating character of V.sub.Ls (
Example 4: Thermostability Measurements by Circular Dichroism (CD) Spectroscopy
(89) To assess whether the additional, engineered disulfide linkage in the mutant V.sub.H and V.sub.L would improve protein stability, the thermal stability via measurement of melting temperature (T.sub.m) by CD spectroscopy was evaluated.
(90) For all V.sub.Hs and mutant V.sub.Ls, a Jasco J-815 spectropolarimeter equipped with a Peltier thermoelectric type temperature control system (Jasco, Easton, Md., USA) was used to carry out experiments. A CD cuvette with a path length of 1 mm was used. The spectra were recorded over a wavelength range of 180-260 nm with scanning speed of 50 nm/min, digital integration time (DIT) of 4 s, a band width of 1 nm, data pitch of 1 nm, and an integration time of 1 s. To measure melting temperature or T.sub.m (Greenfield, 2006a; 2006b), CD spectra were recorded over a temperature range of 30 C. to 96 C. All CD spectra were subtracted from the blank corresponding to buffer spectra. Measurements were performed with concentrations of 50 g/mL V.sub.Hs in 100 mM sodium phosphate buffer, pH 7.4. Heat-induced protein denaturation was monitored at 210 nm for all Cys mutant V.sub.Ls and for HVHAm431, HVHAm431S, and HVHP419S; at 205 nm for HVHAm427, HVHAm427S, HVHAm302, HVHAm302S, HVHB82, HVHP421, HVHP426, HVHP428, HVHP429, HVHP420S, HVHP429S, HVHM81S, HVHP430S, HVHP421S, HVHP426S, HVHP428S, HVHM41, and HVHP414S; at 220 nm for HVHPC235; at 200 nm for HVHPC235S, HVHP430, HVHP413, HVHP423, HVHM81, HVHP419, HVHP420, and HVHB82S; at 202 nm for HVHP44, HVHP414, and HVHP423S; at 208 nm for HVHP44S; and at 209 nm for HVHM41S by changes in ellipticity. The fraction folded (ff) was obtained by a formula as described (Greenfield, 2006a; 2006b):
ff=([].sub.T[].sub.U)/([].sub.F[].sub.U)formula I
where [].sub.T is the molar ellipticity at any temperature, [].sub.F, is the molar ellipticity of the fully folded protein at 30 C. and [O].sub.U is the molar ellipticity of the unfolded protein at 90 C. Melting temperature (T.sub.m) was obtained as a midpoint of the unfolding curve (fraction folded (ff) versus temperature) by a nonlinear regression curve fit (Boltzman sigmoidal equation) using the graphing software GraphPad Prism (version 4.02 for Windows) and were recorded in Table 4.
(91) For wild-type V.sub.L, a Jasco J-810 spectropolarimeter with a NESLAB RTE-111 bath accessory was used. A circular cell with a pathlength of 0.02 cm was used. The spectra were recorded with scanning speed of 100 nm/min, a band width of 1 nm and an integration time of 1 s. The CD spectra were recorded over a temperature range of 25 C. to 85 C. for HVLP342, HVLP351 and HVLP3103, 25 C. to 80 C. for HVLP324, HVLP325, HVLP364 and HVLP389, and 25 C. to 90 C. for HVLP335. Heat-induced changes in molar ellipticity were monitored at 203 nm for HVLP325 and HVLP389 and at 218 nm for HVLP324, HVLP335, HVLP342, HVLP351, HVLP364 and HVLP3103. Measurements were performed in sodium phosphate buffer with concentrations ranging from 4.110.sup.5 M to 5.710.sup.5 M. All CD spectra were subtracted from the blank corresponding buffer spectra and melting temperature (T.sub.m) was obtained as previously described for V.sub.Hs and mutant V.sub.Ls.
(92) TABLE-US-00008 TABLE 4 Affinity constants, K.sub.Ds, and melting temperatures, T.sub.ms, of V.sub.Hs, V.sub.Ls, and corresponding Cys mutants. For V.sub.Ls, the K.sub.Ds are against protein L, for V.sub.Hs against protein A. V.sub.L K.sub.D(M) T.sub.m ( C.) HVLP324 0.2, 0.07.sup.a 59.1 HVLP324S 0.09.sup.a 73.4 HVLP325 1 64.6 HVLP325S 1 82.5 HVLP335 2 57.9 HVLP335S 2 79.0 HVLP342 0.6, 0.04.sup.a 52.6 HVLP342S 0.05.sup.a 63.8 HVLP351 2 54.1 HVLP351S 0.7 71.9 HVLP364 3 52.4 HVLP364S not determined 72.3 HVLP3103 1 55.8 HVLP3103S 1 76.4 HVLP389 1 49.3 HVLP389S 0.4, 1.sup.c 66.3 HVHAm302.sup.b 3.0 52.8 HVHAm302S 10 65.4 HVHAm427.sup.b 1.6 70.9 HVHAm427S 4 84.2.sup.e HVHAm431.sup.b 4 69 HVHAm431S 8 84.6.sup.e HVHPC235 0.3 55.8 HVHP44 1.3.sup.g 64.2 HVHP44S 4.2 74.5 HVHB82 0.2.sup.g 57.9 HVHB82S 1.2 72.9 HVHP421 1.0.sup.g 57.3 HVHP421S 2.8 69.7 HVHP419 1.6.sup.g 56.9 HVHP419S 4.8 (3.2).sup.d 67.4 HVHP430 2.3.sup.g 71.2/72.5.sup.f HVHP430S 7.7 82.7 HVHP429 1.3.sup.g 58.5 HVHP429S 5.5 (3.4).sup.d 71.8 HVHM41 0.5.sup.g 60.8 HVHM41S 1.5 (1.8).sup.d 72.2 HVHM81 1.3.sup.g 65.8/67.9.sup.f HVHM81S 2.1 76.8 HVHP428 1.8.sup.g 62.3 HVHP428S 1.6 73.1 HVHP420 1.2.sup.g 57.8 HVHP420S 2.2 67.3 HVHP414 1.6.sup.g 55.3 HVHP414S 1.8 64.7 HVHP423 3.0.sup.g 55.0 HVHP423S not determined 70.7 HVHP413 0.3.sup.g 54.2 HVHP413S 1.5 not determined HVHP426 0.8.sup.g 62.3/63.1.sup.f HVHP426S 3.4 79.9 HVHP235S 3 72.3 .sup.aThe smaller K.sub.D values correspond to the binding of HVLP324, HVLP324S HVLP342, and HVLP342S to the high affinity sites on protein L. .sup.bSee Table 1 in Arbabi-Ghahroudi et al. (2009b). .sup.c2 very close K.sub.Ds were obtained. .sup.dThe values in the parentheses were obtained with higher concentration of monomer. .sup.eRepresents minimum estimated T.sub.m due to not-so-defined lower plateau (see melting curve profiles in FIG. 5A. .sup.fDone in duplicates. .sup.gK.sub.Ds taken from To et al., 2005.
(93) The melting temperatures (T.sub.ms) of V.sub.Hs (HVHAm302 and HVHAm302S, HVHAm427 and HVHAm427S, HVHAm431 and HVHAm431S, HVHPC235 and HVHPC235S, HVHP44 and HVHP44S, HVHB82 and HVHB82S, HVHP421 and HVHP421S, HVHP419 and HVHP419S, HVHP430 and HVHP430S, HVHP429 and HVHP429S, HVHM41 and HVHM41S, HVHM81 and HVHM81S, HVHP428 and HVHP428S, HVHP420 and HVHP420S, HVHP414 and HVHP414S, HVHP423 and HVHP423S, HVHP413 and HVHP413S, and HVHP426 and HVHP426S) and V.sub.Ls were determined based on ellipticity data assuming a two-state system, which is in agreement with the observed denaturation curves corresponding to a sharp transition into denaturation (
(94) T.sub.ms of V.sub.Ls were in the range of 49.3 C.-64.6 C., a range observed in other sdAbs (Jespers et al., 2004; Tanha et al., 2006) (
Example 5: Surface Plasmon Resonance (SPR)
(95) The protein A and protein L binding property of V.sub.Hs and V.sub.Ls, respectively, was used in SPR binding analyses to probe any subtle possible structural changes due to the engineered disulfide linkages in V.sub.H and V.sub.L mutants. Protein A is often used to monitor conformational integrity of V.sub.Hs (Starovasnik et al., 1999). Standard procedures were carried out for SPR analysis of V.sub.Hs and V.sub.Ls. V.sub.HS and V.sub.Ls were subjected to Superdex 75 10/300 (GL) size exclusion chromatography (GE Healthcare) in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% P20 surfactant) with flow rate of 0.5 mL/min prior to BIACORE analysis and purified monomer peaks collected even in the absence of any evidence of aggregated material. The binding kinetics for the interaction of the V.sub.HS to protein A (Pierce, Nepean, ON, Canada) and V.sub.Ls to protein L (Pierce) were determined by SPR using BIACORE 3000 biosensor system (GE Healthcare).
(96) For the V.sub.HS, HVHAm302, HVHAm302S, HVHAm427, HVHAm427S, HVHAm431 and HVHAm431S, protein A and ovalbumin (as a reference protein) (Sigma, Oakville, ON, Canada) of 540 RUs and 1350 RUs, respectively, were immobilized on research grade CM5 sensor chips (GE Healthcare). Immobilizations were carried out at 50 g/mL in 10 mM acetate buffer, pH 4.5, using the amine coupling kit supplied by the manufacturer (GE Healthcare). All measurements were carried out at 25 C. in HBS-EP buffer at a flow rate of 50 L/min. Surfaces were regenerated by washing with the running buffer.
(97) For HVHPC235 and HVHPC235S, approximately 1,100 RUs and 1,000 RUs of recombinant protein A and ovalbumin (reference protein), respectively, were immobilized on research grade sensor chip CM5. Immobilizations were carried out at 50 g/mL in 10 mM acetate buffer (pH 4.0 or pH 4.5 for protein A or ovalbumin, respectively) using the amine coupling kit supplied by the manufacturer. All measurements were carried out at 25 C. in HBS-EP buffer at a flow rate of 20 L/min or 40 L/min for HVHPC235 or HVHPC235S, respectively.
(98) For mutant V.sub.HS (HVHP44S, HVHB82S, HVHP421S, HVHP419S, HVHP430S, HVHP429S, HVHM81S, HVHP428S, HVHP420S, HVHM41S, HVHP423S, HVHP413S, HVHP426S, and HVHP414S), approximately 1,170 RUs and 1,240 RUs of recombinant protein A and ovalbumin (reference protein), respectively, were immobilized on research grade sensor chip CM5. Immobilizations were carried out at 50 g/mL in 10 mM acetate buffer (pH 4.5 for both protein A and ovalbumin) using the amine coupling kit supplied by the manufacturer. All measurements were carried out at 25 C. in HBS-EP buffer at a flow rate of 20 L/min or 40 L/min for HVHB82S, HVHP421S, HVHP429S, HVHM41S, HVHM81S, HVHP423S, HVHP413S, HVHP426S, and HVHP414S or HVHP44S, HVHP419S, HVHP430S, HVHP428S, and HVHP420S, respectively. The protein A binding property of the wild-type counterparts were determined previously (To et al 2005).
(99) For wild-type V.sub.Ls, 600 RUs of protein L or 800 RUs of a Fab reference protein were immobilized on research grade CM5 sensor chips (GE Healthcare). For V.sub.L mutants, approximately 400 RUs of recombinant protein L or ovalbumin (reference protein) were immobilized. Immobilizations were carried out at protein concentrations of 20 or 50 g/mL in 10 mM acetate buffer, pH 4.5, using the amine coupling kit supplied by the manufacturer (GE Healthcare). All measurements were carried out at 25 C. in HBS-EP buffer at a flow rate of 40 or 50 L/min. Surfaces were regenerated by washing with the running buffer. All data were evaluated using the BIAevaluation 4.1 software (GE Healthcare).
(100) The SPR analysis showed that the vast majority of mutant V.sub.Hs (HVHAm302S, HVHAm427S, HVHAm431S, HVHPC235S, HVHB82S, HVHP421S, HVHP419S, HVHP430S, HVHP429S, HVHM81S, HVHP420S, HVHM41S, HVHP423S, HVHP413S, HVHP426S, and HVHP44S) bound to protein A with less affinity, reflected by higher K.sub.D values, compared to their wild-type counterparts (
(101) It was found that the V.sub.L Cys mutants bound to protein L with affinities similar to their wild-type counterparts (
Example 6: Protease Stability
(102) The effect of the engineered disulfide linkage on the protease stability of V.sub.Ls and V.sub.Hs was evaluated using major GI proteases trypsin, chymotrypsin and pepsin. After the digestion reactions with GI proteases, V.sub.Ls and V.sub.Hs were examined for the occurrence of enzymatic cleavage by SDS-PAGE and mass spectrometry. Digestions were performed essentially as described (Hussack et al., 2011).
(103) For V.sub.Ls, digestion experiments were performed in a total volume of 304 with 6 g of V.sub.L at 37 C. for 1 h with an enzyme to sdAb ratio of 1:200, 1:40, 1:20, and 1:10 (trypsin/chymotrypsin), or 1:200, 1:20, 1:10, and 1:4 (pepsin). Tryptic and chymotryptic digestion experiments were carried out using sequencing grade enzymes (Hoffmann-La Roche Ltd., Mississauga, ON, Canada), according to the manufacturer's instructions. Pepsin (Sigma) digestion reactions were performed at pH 2.0 where the pH was adjusted by adding appropriate volume of 400 mM HCl. In control experiments, enzymes were replaced with equal volume of reaction buffer. Reactions were stopped by adding an equal volume of SDS-PAGE sample buffer (containing 0.2 M dithiothreitol) and boiling the mixture at 95 C. for 5 min. Samples were subsequently subjected to analysis by SDS-PAGE and the percent of intact sdAbs after protease digestions were determined by spot density analysis as described in Hussack et al., In press(a) and compared with controls to calculate the percentage of V.sub.Ls with intact structure after protease digestion.
(104) For V.sub.Hs, digestion experiments with trypsin/chymotrypsin/pepsin were performed in a total volume of 30 L with 6 g of V.sub.H and its mutant V.sub.H counterpart in parallel at 37 C. for 1 h with an enzyme to sdAb ratio of 1:20 and duplicated by two independent trials. The digestions and analyses were carried out as described for the V.sub.Ls.
(105) The results are shown in
(106) The correlation between the thermal stability and the protease resistance was further explored by plotting protease resistance (%) vs. T.sub.m (
(107) In the case of the V.sub.HS that are shown in Table 5, the mutant V.sub.HS showed higher resistance (% median) to all the three GI proteases (77% and 82% [trypsin], 69% and 75% [chymotrypsin], and 96% and 98% [pepsin], for the wild-type and the mutant V.sub.HS, respectively), although the differences were not that significant (Wilcoxon matched pairs t test, two-tailed P values=0.0899 [trypsin], 0.7896 [chymotrypsin]), and 0.7832 [pepsin]). While most of the mutant V.sub.HS showed unchanged or slightly improved resistance against pepsin, two mutant V.sub.HS (HVHM81S and HVHP423S) showed decreased resistance from 100% (for both HVHM81 and HVHP423) to 53% (for HVHM81S) and to 68% (for HVHP423S), although the pepsin resistance of HVHM81S seemed to be underestimated due to inability of the spot density analysis tool to recognize the protein bands properly. Intriguingly, large improvements in terms of trypsin resistance were shown from two mutant V.sub.HS (HVHP44S and HVHP413S) where their trypsin resistance improved from 5% and 29% (HVHP44 and HVHP413, respectively) to 57% and 100% (HVHP44S and HVHP413S, respectively). Taken together, the mutants are as resistant to proteases as the wild type counterparts.
(108) TABLE-US-00009 TABLE 5 GI protease resistance of V.sub.Hs Trypsin Chymotrypsin V.sub.H Resistance (%) Resistance (%) Pepsin Resistance (%) HVHP44 5 2 69 1 95 9 HVHP44S 57 2 63 7 100 10 HVHB82 92 5 100 19 100 16 HVHB82S 87 7 64 17 95 28 HVHP421 74 7 59 7 97 25 HVHP421S 100 10 78 31 100 22 HVHP419 72 7 52 2 87 11 HVHP419S 75 3 52 1 85 6 HVHP430 95 8 100 19 100 3 HVHP430S 100 19 74 24 100 3 HVHP429 89 6 70 7 95 28 HVHP429S 82 12 86 2 100 5 HVHM41 11 3 96 32 100 16 HVHM41S 22 18 68 13 93 16 HVHM81 86 4 95 8 100 4 HVHM81S 75 1 95 6 .sup.53 3.sup.a HVHP428 92 1 65 2 87 1 HVHP428S 98 4 82 3 90 13 HVHP420 80 12 52 0 77 2 HVHP420S 100 14 52 10 100 2 HVHP414 72 6 35 3 9 7 HVHP414S 74 1 49 9 57 15 HVHP423 61 6 88 21 100 15 HVHP423S 82 15 97 29 68 30 HVHP413 29 63 100 HVHP413S 100 76 100 HVHP426 84 3 69 2 85 6 HVHP426S 76 3 97 15 100 1 .sup.aThe pepsin resistance is an under-estimation.
Example 7: Sequence Identities Between VHs and Between VLs
(109) Sequence identities between the V.sub.Hs, as well as V.sub.Ls were determined. Only FR sequences were included in the analysis, i.e., CDR sequences were excluded.
(110) The sequences of V.sub.H pairs or V.sub.L pairs were aligned using ClustalW (Thompson et al, 1994), and the percentage identity between the V.sub.H pairs or V.sub.L pairs was calculated using the BioEdit Sequence Alignment Editor.
(111) The biophysical consequences of Cys pair substitutions at positions 49 and 69 for V.sub.Hs and 48 and 64 for V.sub.Ls are shown to be universal irrespective of their sequence, germline origin, and CDR length and composition. The V.sub.Hs have diverse germline origin and CDR lengths and amino acid composition. In particular, CDR3 length and composition is more diverse. Nonetheless, independent of these structural variations, the introduction of Cys pairs at positions 49 and 69, leads to the formation of Cys49/Cys69 disulfide linkage and significant improvements in non-aggregation, thermal stability and protease stability of V.sub.Hs without compromising their expression yields. The 32 V.sub.Ls described in WO 2006/099747 are diverse: they belong to both kappa and lambda families and have diverse V segment and J segment germline origin and CDR amino acid compositions and lengths. The length of CDR3, in particular ranges from 6 to 11 amino acids. 8 non-aggregating V.sub.Ls representing different classes (
Example 8: Construction of a Disulfide-stabilized VL Phage Display Library
(112) The HVLP324S V.sub.L phage display library was constructed in two steps by the method described by Hussack et al. (In press (c)). First, a library with a randomized CDR3 was constructed based on the HVLP324S V.sub.L scaffold (
(113) (i) Construction of the CDR3-Randomized V.sub.L Phage Display Library
(114) Phage ssDNA containing uridine instead of thymidine (dU-ssDNA template) was prepared as described (Hussack et al. In press (c)), except that the phage used for ssDNA preparation was fd-tetV.sub.L24S. fd-tetV.sub.L24S is fd-tetGIIID phage (Tanha et al., 2001) with the HVLP324S V.sub.L gene in its genome and fused with the phage p3 gene (
(115) (ii) Construction of the CDR1/CDR2/CDR3-Randomized V.sub.L Phage Display Library
(116) In the second step of the library construction, CDR1 and CDR2 were randomized. dU-ssDNA template was prepared starting with 410.sup.12 cfu CDR3-randomized library phage from above (see Hussack et al (c)). 200 g of ssDNA was obtained from 2 mL of phage (210.sup.9/L). Incorporation of uridine into phage ssDNA was confirmed as above. The dU-ssDNA template was used in the second round of mutagenesis which used mutagenic oligonucleotides V.sub.L24-CDR1 and V.sub.L24S-CDR2. Annealing and CCC-DNA synthesis steps were essentially identical to those described above. A total of 300 g CCC-DNA in 1 mL ddH.sub.2O was prepared. Seventy-seven transformations (13 L CCC-DNA plus 200 L of electrocompetent E. coli TG1) were carried out as described. Following incubation in SOC medium, transformation materials were titrated and grown in 3 L of 2YT/tetracycline (5 g/mL) overnight at 180 RPM and 37 C. Frozen library stocks were made and its cell density was estimated as described above. The library phage was purified from the supernatant in a final volume of 20 mL PBS, titred against TG1, and stored frozen at 80 C. in 500 L aliquots for future first round panning as the input phage. The library size was determined to be 110.sup.8 transformants. 78 V.sub.L clones from the library (non-amplified library) titer plates were subjected to sequencing as described in Hussack et al (c).
(117) HVLP324S V.sub.L was used as the scaffold for construction of a disulfide-stabilized synthetic V.sub.L library. HVLP324S is the mutant version of the previously described HVLP324 but with an additional disulfide linkage between Cys48 and Cys64. The HVLP324S V.sub.L phage display library was constructed by introducing diversity at 16 (CDR3=9 amino acids), 17 (CDR3=10 amino acids), and 18 (CDR3=11 amino acids) locations in all 3 CDRs essentially as described for a previously described HVLP324 V.sub.L phage display library (
(118) A CDR3 length distribution analysis of the 76 library clones showed that the majority (71%) of the library clones had a CDR3 length of 9 amino acids. Clones with CDR3 lengths of 10 and 11 amino acids were at 18% and 11%, respectively.
(119) Additional details of the library design, construction and characterization are provided in the legend and figure for
(120) TABLE-US-00010 TABLE6 Listofoligonucleotidesusedformolecularcloning. Name Sequence(5>3) Purpose SEQID V.sub.L24-CDR1 AGAGTCACCATCACTTGCNNKGCAAGT Mutagenesis 87 CAGRGCATTNNKNNKNNKTTANNKTGG TATCAGCAGAAACCA V.sub.L24S-CDR2 CCTAAACTCCTGTGCTTTNNKGCATCC Mutagenesis 88 NNKCKNNNKAGTGGGGTCCCATCAAGG V.sub.L24-CDR3 TTTGCAACTTACTACTGTNNKCAGNNK Mutagenesis 89 NNKNNKNNKCCTNNKACGTTCGGCCAC GGGACC V.sub.L24-CDR3a TTTGCAACTTACTACTGTNNKCAGNNK Mutagenesis 90 NNKNNKNNKCCTNNKNNKACGTTCGGC CACGGGACC V.sub.L24-CDR3b TTTGCAACTTACTACTGTNNKCAGNNK Mutagenesis 91 NNKNNKNNKCCTNNKNNKNNKACGTTC GGCCACGGGACC 96GIII CCCTCATAGTTAGCGTAACGATCT Colony-PCR 92 Sequencing fdTGIII GTGAAAAAATTATTATTCGCAATTCCT Colony-PCR 93 Sequencing HV.sub.L24-BamHI TTGTTCGGATCCTAGGACGGTCACCT Subcloning 94 HV.sub.L24-BbsI TATGAAGACACCAGGCCGACATCCAG Subcloning 95 KT124 TGTGCTACCACCACTACTACTAATAGC Mutagenesis 96 GCAGACCCACTCCAGCCCCTTCCCTGG KT125 CACGGTGTTCTTGGAATTGTCTCTGGA Mutagenesis 97 GCAGGTGAATCGGCCCTTCACGGAGTC M13RPb CAGGAAACAGCTATGAC Colony-PCR 98 Sequencing N: A, T, G, or C. K: T or G. R: A or G.
Example 9: Validation of the Disulfide-Stabilized VL Phage Display Library
(121) To validate HVLP324S V.sub.L phage display library, the library was subjected to selection for binders to two different test antigens, namely human lysozyme and human serum albumin (HSA). Positive candidate binders (phage-displayed V.sub.Ls) identified by initial binding assays (phage ELISA) were subcloned in vectors for expression in E. coli. V.sub.L binders were expressed, purified and analyzed for affinity and stability.
Example 9a: Panning
(122) Selection for anti-human lysozyme V.sub.Ls was performed by standard panning techniques (see for example, Lee et al., 2007; Arbabi-Ghahroudi et al., 2009a). Briefly, a Nunc microtiter well (VWR International, Ltd., Mississauga, ON, Canada) was coated with 100 L of 1 mg/mL human lysozyme (Sigma-Aldrich, Mississauga, ON, Canada) in sterile PBS (3.2 mM Na.sub.2HPO.sub.4, 0.5 mM KH.sub.2PO.sub.4, 1.3 mM KCl, 135 mM NaCl, pH 7.4) overnight at 4 C. The well was washed 3 with sterile PBS, blotted on a paper towel, and blocked with 150 L of 2% (w/v) milk in sterile PBS (2% MPBS) at 37 C. for 2 h. The blocking agent was removed from the well and 100 L of library phage (amplified phage prepared in Example 8, Section (ii) in 1% MPBS (510.sup.11 cfu; preincubated at 37 C. for 1.5 h) were added to the well and incubated for 1.5 h at 37 C. After 10 washes with PBST (0.05% (v/v) Tween-20 in sterile PBS), phage were eluted by incubating the well with 100 L of freshly prepared 124 mM triethylamine for 10 min at room temperature, pipetting up and down at 8 min. The eluted phage solution was neutralized in a 1.5 mL microtube by mixing with 50 L of 1 M Tris-HCl, pH 7.5 and kept on ice until the infection phase. 25 mL of exponentially growing E. coli TG1 cells (OD.sub.600=0.5) was prepared (Arbabi-Ghahroudi et al., 2009a) in 50 mL sterile Falcon tubes from which 2 mL was infected at 37 C. for 30 min with 145 L of eluted phage (5 L was kept aside for determining the titer of the eluted phage as described below). The infected cells were centrifuged at 4,000 rpm and 4 C. for 20 min using Sorval Legend RT rotor and centrifuge (Thermo Fisher Scientific, Nepean, ON, Canada), the supernatant was removed, and the pellet was resuspended in 1 mL of 2YT and equally spread onto 3 large Petri dishes with 2YT agar/5 g/mL tetracycline (2YT/Tet). The Petri dishes were incubated at 37 C. overnight. Next day, the amplified phage were recovered from the large Petri dishes. Briefly, 10-15 mL of sterile PBS (volume added according to the colony density) was added to each plate, the cells were scraped off using a glass spreader, and collected in sterile 50 mL sterile Falcon tubes. The cells were scraped off once more as before and pooled with the previous ones. The suspension was fractionated into cell pellet and supernatant by centrifugation for 20 min at 4,000 rpm and 4 C. using Sorval Legend RT rotor and centrifuge (Thermo Fisher Scientific). The amplified phage were purified from the supernatant essentially as described (Lee et al., 2007). Briefly, to the phage supernatant that was prefiltrated with sterile 0.22 m filter was added a 1/5 volume of 20% PEG-8000/2.5 M NaCl in 50 mL sterile Falcon tubes, and the mixture was incubated on ice for 1 h. The solution was centrifuged for 20 min at 4,000 rpm and 4 C. using Sorval Legend RT rotor and centrifuge (Thermo Fisher Scientific). The phage pellet was redissolved in 3 mL of sterile PBS, transferred to a 15-mL sterile Falcon tube, mixed with 1/5 volume of 20% PEG-8000/2.5 M NaCl (0.6 mL), and incubated on ice for 20 min. The phage solution was centrifuged at 4,000 rpm and 4 C. for 20 min using Sorval Legend RT rotor and centrifuge (Thermo Fisher Scientific), and the phage pellet was dissolved in 0.5-1 mL sterile PBS, dissolution volume depending on the size of the pellet. The phage solution was aliquoted in 0.1 mL volumes in a sterile 1.5-mL sterile microtube. The titer of the amplified phage was determined as described below.
(123) For the second round of panning, in parallel with the conventional panning described above, thermopanning was also performed where the amplified phage in sterile PBS from the first round was heated at 65 C. for 2 h in a 1.5 mL sterile microtube, subsequently centrifuged, and the phage supernatant was added to the human lysozyme-coated well for binding (in the second round of panning). All the remaining steps were the same for both methods of panning. Parallel pannings were continued for two more rounds. Washes were 12, 15, and 15 for the second, third and fourth rounds of panning, respectively.
(124) Panning (conventional panning) against human serum albumin was performed as described for human lysozyme except that no thermopanning in parallel was performed. Following panning, standard phage ELISA (Harrison et al., 1996; Tanha et al., 2001; Hussack et al., In press (c)) was performed on clones from titer plates (obtained from eluted phage) to identify phage displaying V.sub.Ls positive for binding to human lysozyme or serum albumin.
Example 9b: Phage Titer Determination
(125) Standard techniques were used to determine phage titers. To determine the titer of the eluted phage, dilutions of the eluted phage were made in PBS at 10.sup.1-10.sup.4 (2L of phage was added to 18 L of PBS to make the 10.sup.1 dilution and so on). 10 L of each dilution was mixed with 100 L of exponentially growing E. coli TG1 cells (OD.sub.600=0.5), and the mixtures were incubated at 37 C. for 30 min for phage infection of E. coli TG1 cells to occur. The infected TG1 cells were plated on 2YT/Tet agar Petri dishes and incubated at 37 C. overnight. The number of colonies was used to determine the titer (cfu/mL) of the eluted phage. Colony-PCR on colonies was performed to verify the presence of inserts (positive hits gave a size of around 500 bp) and to provide templates for DNA sequencing using fdTGIII and 96GIII primers (Table 6). Sequences were analyzed and manipulated by DNASTAR Lasergene 8.
(126) Briefly, to determine the titer of the amplified phage (input phage), phage dilutions were made at 10.sup.3, 10.sup.6, 10.sup.8, and 10.sup.10. 10 L of each diluted phage was mixed with 100 L of exponentially growing TG1 cells, and the mixtures were incubated at 37 C. for 30 min for phage infection of cells to occur. Subsequently, using sterile disposable spreaders, the phage infected cells were spread on Petri dishes with 2YT r/Tet agar media and left in clean bench for 5 min. Plates were incubated overnight at 37 C. In the morning, the colony titers on the plates were used to determine the titer of the amplified phage.
(127) To show the utility of the library, the library was panned against human lysozyme and human serum albumin for four rounds. In the instance of the panning against human lysozyme, a conventional panning was performed in the first round followed by conventional panning and thermopanning in parallel from the 2.sup.nd round on. In the thermopanning approach, the input phage was incubated at 65 C. for 2 h, centrifuged to remove any possible aggregates, and the supernatant was added to the lysozyme-coated well for binding. In the conventional panning approach, the input (amplified) phage was directly added to the lysozyme-coated well without the heat treatment. The thermopanning was applied to see if it preferentially enriches for stable (thermostable and/or non-aggregating) V.sub.L binders compared to the conventional panning which was expected to be indiscriminate with regards to stable and unstable V.sub.L binders.
(128) Table 7 shows the results of panning against human lysozyme with respect to the output phage titer (or eluted phage titer). In general, the output phage titers are 3-4 orders of magnitude lower for the thermopanning approach compared to the conventional one. This may be due to lower input phage titers used and further loss of phage (possibly aggregating phage-displayed V.sub.Ls) to aggregation during the heat treatment (data not shown).
(129) TABLE-US-00011 TABLE 7 Phage titers for pannings performed under room temperature (conventional panning) versus 65 C.-2 h (thermopanning) selection conditions. Data are for panning against human lysozyme. cfu: colony-forming units. Room temperature 65 C.-2 h Panning round Input (cfu) output (cfu) Input (cfu) output (cfu) 1 5 10.sup.11 7 10.sup.4 N/A N/A 2 4 10.sup.10 2 10.sup.5 4 10.sup.10 2 10.sup.2 3 7 10.sup.11 2 10.sup.5 2 10.sup.10 6 10.sup.4 4 5 10.sup.11 6 10.sup.7 5 10.sup.10 7 10.sup.4
(130) Phage ELISA and sequence analyses from round 3 and 4 identified 9 anti-human lysozyme V.sub.Ls with variable affinities to immobilized human lysozyme (
(131) TABLE-US-00012 TABLE8 Propertiesofanti-humanlysozymeandanti-human serumalbuminVLSisolatedfromrounds3and4ofpanning. V.sub.L K.sub.D(M) T.sub.m(C.) Monomer/Agg. Selection HVLHLRN(SEQIDNO:101) 0.05 74 Monomer 65C.-2h HVLHLDS(SEQIDNO:100) 6.1 68.3 Monomer 65C.-2h HVLHLEM(SEQIDNO:104) >73 72.2 Monomer RT HVLHLAQ(SEQIDNO:103) 0.2 66.5 Agg.(7%) RT HVLHLQI(SEQIDNO:105) 2.6 62 Agg.(17%) RT HVLHLNE(SEQIDNO:102) 0.06 62 Agg.(13%) RT/65C.-2h HVLHLYS(SEQIDNO:106) 6.4 66.8 Agg.(7%) RT HVLHLNN(SEQIDNO:99) 0.2 62.6 Agg.(12%) RT/65C.-2h HVLHLFL(SEQIDNO:108 ND ND ND RT/65C.-2h HVLHSA1(SEQIDNO:107) 0.8 71 Monomer RT ND, not determined; RT, room temperature Agg., aggregate. %s are % aggregates.
(132) Following 4 rounds of panning against human serum albumin, screening of 50 clones by phage ELISA and sequencing revealed one predominant V.sub.L, namely, HVLHSA1 (
(133) All the 10 anti-lysozyme and anti-albumin phage-V.sub.L binders were subsequently processed for further characterization as described below.
Example 9c: Biophysical Characterization of Anti-Human Lysozyme and Anti-Human Serum Albumin VLs
(134) Starting with phage-displayed V.sub.L clones, V.sub.Ls were cloned, expressed, and purified by standard techniques as described above for other antibody domains. The V.sub.Ls were subsequently analyzed by size exclusion chromatography for aggregation status, by CD for thermostability, and by surface plasmon resonance for their binding affinity and specificity.
(135) Nine anti-human lysozyme V.sub.Ls and HVLHSA1 V.sub.L (
(136) Subsequently, V.sub.Ls were analyzed for their aggregation behaviour by Superdex 75 size exclusion chromatography. % aggregate (or % monomer) for each V.sub.L was also determined as described (Arbabi-Ghahroudi et al. 2010). As shown in
(137) To obtain a measure of the thermostability of the V.sub.L binders, melting temperatures (T.sub.ms) of the V.sub.Ls were determined by CD as described previously for other domains.
(138) The affinities (K.sub.Ds) of the anti-human lysozyme V.sub.Ls to their antigen, human lysozyme, was also determined. The binding of human lysozyme to immobilized anti-human lysozyme V.sub.L binders was determined by surface plasmon resonance (SPR) using BIACORE 3000 (GE Healthcare). 298 resonance units (RUs), 312 RUs, 295 RUs, 339 RUs, 315 RUs, 412 RUs, 370 RUs, 345 RUs, and 349 RUs of each V.sub.L binders (HVLHLAQ, HVLHLNE, HVLHLEM, HVLHLYG, HVLHLRN, HVLHLNN, HVLHLQI, HVLHLYS, and HVLHLDS, respectively) was immobilized on a research grade CM5 sensor chip. Immobilizations were carried out at concentrations of 10 g/mL in 10 mM acetate at pH 4.5 using the amine coupling kit supplied by the manufacturer. Human lysozyme was passed through a Superdex 75 column (GE Healthcare) in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% P20 surfactant) with flow rate of 0.5 mL/min to eliminate any possible aggregates prior to Biacore analysis. For the binding studies, analyses were carried out at 25 C. in 10 mM HEPES, pH 7.4 containing 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20. The flow rates used were 20 L/min. For regeneration the surfaces were washed for 15 min with the running buffer. Data were analyzed with BIAevaluation 4.1 software. Binding profiles and calculated K.sub.Ds are shown in
(139) The K.sub.D of the V.sub.L against human serum albumin was determined by surface plasmon resonance as described for other domains above. Briefly, the binding of the V.sub.L to immobilized human serum albumin was determined by SPR using the BIACORE 3000 (GE Healthcare). Approximately 1,600 RUs of human serum albumin, 1,900 RUs of bovine serum albumin and 2,200 RUs of ovalbumin were immobilized on a research grade CM5 sensor chip. An ethanolamine blocked surface was prepared, as a reference, in exactly the same manner as the protein surfaces. Immobilizations were carried out at concentrations of 20 g/mL in 10 mM acetate buffer pH 4.5 using the amine coupling kit supplied by the manufacturer. The V.sub.L was passed through a Superdex 75 column (GE Healthcare) to eliminate any possible aggregates prior to Biacore analysis. For the binding studies, analyses were carried out at 25 C. in 10 mM HEPES, pH 7.4 containing 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20. The flow rates used were 40 L/min and sample volumes were 60 L. For regeneration the surface was washed for 10 min with the running buffer. Data were analyzed with BIAevaluation 4.1 software.
(140) Thus the results show that V.sub.H and V.sub.L domains with non-canonical disulfide bonds are feasible as scaffolds for constructing V.sub.H/V.sub.L synthetic libraries that are sources of antigen binders with high affinity, stability (non-aggregation and thermostability), and expression yields.
Example 10: Construction of a Disulfide-Stabilized VH Phage Display Library
(141) Experiments were designed to create a V.sub.H phage display library with randomized CDRs built on a V.sub.H scaffold stabilized by extra disulfide linkage at positions 49 and 69. To construct the library, a V.sub.H phage display library (HVHP430LGH3) was used as a template to introduce additional disulfide linkage by replacing two selected amino acid residues (49Ser and 69Ile) with cysteines using the method described above for the V.sub.L library and previously (Sidhu et al., 2000; Hussak et al (c)). The HVHP430LGH3phage display library is a phagemid vector-based library with the CDR-randomized human V.sub.H genes cloned into the phagemid pMED-1 and fused with protein 3 gene. (Non-aggregating human V.sub.H domains PCT publication WO2009/079093).
(142) The procedure to construct the disulfide-stabilized V.sub.H phage display library was performed essentially as described above and in Hussack et al (c) and Sidhu et al., 2000). Briefly, the construction was done by two separate rounds of site-directed mutagenesis on the two amino acid residues selected to be replaced with cysteines. Firstly, dU-ssDNA (uridine-containing single strand phage DNA) was prepared using E. coli CJ236 infected with phage (410.sup.12 cfu (colony-forming unit)) from HVHP430LGH3 library. After confirming the incorporation of uridine into the dU-ssDNA by determining titers against E. coli TG1 and CJ236 as described above and in Hussack et al (c), a series of in vitro DNA synthesis reaction including phosphorylation of the primer IT, for annealing to mutagenize 49Ser to 49Cys, ligation, and polymerization was subsequently performed using 20 g of the dU-ssDNA. The in vitro DNA synthesis reaction resulted in 23 g of heteroduplex DNA in total. Subsequent 10 separate transformations using 50 L of E. coli TG1 competent cells with 564 ng of the heteroduplex DNA for each transformation gave the diversity of 1.110.sup.10 cfu in total. Clonal selections by PCR with primers (M13RPa or b and 96GIII) revealed that about 50% (8 out of 16) of the clones had the 49Ser to 49Cys mutation. For the next round of mutagenesis, phage was prepared from the 49Ser to 49Cys mutated phagemid library ((HVHP430LGH3C1)) by rescuing them with M13K07 helper phage, resulting in the phage titer of 2.110.sup.13 cfu/mL. dU-ssDNA was subsequently prepared from E. coli CJ236 using the phage (410.sup.12 cfu) from (HVHP430LGH3C1) which was followed by in vitro DNA synthesis reaction with 8 g of the dU-ssDNA and the primer KT125 annealing to mutagenize another amino acid residue (69Ile to 69Cys), resulting in 12 g of heteroduplex DNA in total. Subsequent 22 separate transformations using 50 L of E. coli TG1 competent cells with 230 ng of the heteroduplex DNA for each transformation gave the library size of 5.310.sup.9 cfu in total. To determine the percentage of the library that represents V.sub.H genes carrying double mutations (49Ser to 49Cys and 69Ile to 69Cys), 65 colonies were screened by PCR with primers (M13RP a or b and 96GIII), sequenced and analyzed, revealing that 28% (18 out of 65) of the clones had double mutations (49Ser to 49Cys and 69Ile to 69Cys), 35% (23 out of 65) had single mutations (49Ser to 49Cys), 15% (10 out of 65) had the single mutation 69Ile to 69Cys, and 22% (14 out of 65) had no mutations. Thus, the size of the library with both Cys mutations (HVHP430LGH3C2) was corrected to 1.510.sup.9 cfu (=5.310.sup.928%) according to the result of the clonal selection. The HVHP430LGH3C2 phage display library was amplified in 300 mL of 2YT, the library cells were resuspended in 15 mL of 10% glycerol, and subsequently kept as 1 mL aliquots at 80 C.
(143) The diversity of the library is high (5.310.sup.9 transformants) with the additional stabilizing 49Ser to 49Cys and 69Ile to 69Cys mutations in all its V.sub.H members. The library should yield binders with high stability to target antigens, due to the presence of Cys49/Cys69 disulfide linkage in its V.sub.H members, using the standard selection and panning techniques known in the art.
(144) Non-Limiting Embodiments of the Invention
(145) Embodiment 1 comprises a composition comprising a immunoglobulin scaffold comprising one or more than one non-canonical disulfide bond in the framework region (FR). Embodiment 1 further comprises a composition comprising a mutant immunoglobulin scaffold comprising one or more than one non-canonical disulfide bond in the framework region (FR), wherein the composition has one or more improvements selected from increased stability, increased melting temperature, increased solubility and decreased aggregation. The improvements of a mutant composition of Embodiment 1 may be selected from improved thermal refolding efficiency, increased resistance to protease digestion, increased shelf life at or above room temperature, at 4 C., or below 0 C. The improvements of a mutant composition of Embodiment 1 may be further characterized as increased or prolonged functionality when administered to a human subject when compared to a corresponding composition that does not comprise the non-canonical disulfide bond.
(146) Embodiment 2 comprises the composition of embodiment 1, wherein the immunoglobulin scaffold is selected from a V.sub.H or a V.sub.L scaffold, wherein the V.sub.H scaffold comprises at least one non-canonical disulfide bond in the FR, wherein the non-canonical disulfide bond is formed between Cys residues introduced at positions 49 and 69, based on Kabat numbering; and the V.sub.L scaffold comprises at least one non-canonical disulfide bond in the FR, wherein the non-canonical disulfide bond is formed between Cys residues introduced at positions 48 and 64, based on Kabat numbering.
(147) Embodiment 3 comprises the composition of embodiment 1 wherein the scaffold is part of a larger antibody protein or fragment selected from the group consisting of a single domain antibody (sdAb), scFv, Fab, F(ab).sub.2, or mature immunoglobulin. Embodiment 4 comprises the composition of embodiment 3 wherein the mature immunoglobulin is selected from the group consisting of an IgG1, IgG2, IgG3, IgG4, IgE, and/or IgM.
(148) Embodiment 5 comprises the composition of embodiment 1 wherein the immunoglobulin scaffold is a V.sub.H. Embodiment 5 further comprises the composition of embodiment 1 wherein the immunoglobulin scaffold is selected from the V.sub.H1 family, V.sub.H2 family or V.sub.H3 family. Embodiment 6 comprises the composition of embodiment 5 wherein the V.sub.H is of the V.sub.H3 family. In a further embodiment, the V.sub.H immunoglobulin scaffold is selected from one or more sequences of the group consisting of SEQ ID NOS: 2, 4, 6, 8, and 70-83, or a sequence substantially identical thereto. In one embodiment, the substantially identical sequence differs by at least 1, at least 2, at least 3, at least 4 or at least 5 residues compared to the disclosed sequence.
(149) Embodiment 7 comprises the composition of embodiment 1 wherein the immunoglobulin scaffold is a V.sub.L. Embodiment 8 comprises the composition of embodiment 7 wherein the V.sub.L is of the kappa or lambda family. In a further embodiment, the V.sub.L immunoglobulin scaffold is selected from one or more sequences of the group consisting of SEQ ID NOS: 10, 12, 14, 16, 18, 20, 22 and 24, or a sequence substantially identical thereto. In one embodiment, the substantially identical sequence differs by at least 1, at least 2, at least 3, at least 4 or at least 5 residues compared to the disclosed sequence.
(150) Embodiment 9 comprises the composition of any one of embodiments 1 to 8, wherein the one or more than one non-canonical disulfide bond is formed between cysteines introduced by mutations into FR2 and FR3. Embodiment 10 comprises the composition of embodiment 5 or 6, wherein the Cys residues are introduced at positions 49 and 69, based on Kabat numbering. Embodiment 11 comprises the composition of embodiment 7 or 8, wherein the Cys residues are introduced at positions 48 and 64, based on Kabat numbering.
(151) Embodiment 12 comprises the composition of any one of embodiments 1 to 11 wherein the scaffolds are multimerized. In one embodiment, the resulting multimer from embodiment 12 comprises at least a dimer, a trimer, tetramer, pentamer, hexamer, heptamer or an octamer.
(152) Embodiment 13 comprises the composition of any one of embodiments 1 to 12 wherein the scaffold is fused to a second sequence, wherein the second sequence is selected from an Fc fragment, a targeting sequence, a signal sequence and a purification tag, or any combination thereof. In one embodiment, at least one unit of the second sequence is fused to at least one monomer comprising the scaffold of the invention In one embodiment, the at least one unit of a second sequence is fused to a composition of embodiment 12.
(153) Embodiment 14 comprises the composition scaffold of any one of embodiments 1 to 13, wherein the immunoglobulin scaffold comprises the framework regions of sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 70-83, or sequences substantially identical thereto, or fragments thereof, with the proviso that the substantially identical sequences retain both the canonical and non-canonical disulfide bonds and wherein the residues comprising CDR1, CDR2 and CDR3 may be any suitable sequence. In a specific embodiment, the composition comprises SEQ ID NO:2 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:4 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:6 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:8 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:10 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:12 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:14 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:16 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:18 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:20 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:22 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:24 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:70 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:71 or a functional fragment thereof.
(154) In a specific embodiment, the composition comprises SEQ ID NO:72 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:73 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:74 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:75 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:76 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:77 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:78 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:79 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:80 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:81 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:82 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:83 or a functional fragment thereof. In a specific embodiment, the composition comprises SEQ ID NO:84 or a functional fragment thereof.
(155) Embodiment 15 comprises a nucleic acid encoding a composition of any one of embodiments 1 to 14. Embodiment 16 comprises an expression vector comprising the nucleic acid of embodiment 15. Embodiment 17 comprises a host cell or organism comprising the expression vector of embodiment 16. In one embodiment, the nucleic acid sequence encoding the immunoglobulin scaffold of the invention encodes a sequence substantially identical to the disclosed compositions of the invention. In one embodiment, the substantially identical sequence differs by at least 1, at least 2, at least 3, at least 4 or at least 5 residues compared to the disclosed sequence. In one embodiment the nucleic acid is codon optimized for expression in a host cell or organism. In one embodiment the nucleic acid is codon optimized for expression in a bacterium. In one embodiment the nucleic acid is codon optimized for expression in E. coli. In one embodiment the nucleic acid is codon optimized for expression in yeast. In one embodiment the nucleic acid is codon optimized for expression in a nonhuman animal cell. In one embodiment the nucleic acid is codon optimized for expression in a mammalian cell. In one embodiment the nucleic acid is codon optimized for expression in a human cell.
(156) Embodiment 18 comprises a recombinant library comprising at least one immunoglobulin scaffold of embodiment 2, wherein the immunoglobulin scaffold further comprises a multiplicity of suitable CDR sequences, wherein the multiplicity of CDR sequences provide a diversity of at least 10.sup.5, at least 10.sup.6, at least 10.sup.7, at least 10.sup.8, at least 10.sup.9 or more than 10.sup.9 clones per library.
(157) Embodiment 19 comprises a method of improving stability of a composition comprising an immunoglobulin scaffold, the method comprising introducing one or more than one non-canonical disulfide bond in the framework region. The method of embodiment 19 further comprises the method of using recombinant technology to replace a first codon and a second codon in an immunoglobulin sequence with a codon for cysteine at each position, wherein the immunoglobulin sequence is a V.sub.H scaffold, and wherein the first codon corresponds to position 49 using Kabat numbering and the second codon corresponds to position 69 using Kabat numbering. The method of embodiment 19 further comprises the method of using recombinant technology to replace a first codon and a second codon in an immunoglobulin sequence with a codon for cysteine at each position, wherein the immunoglobulin sequence is a V.sub.L scaffold, and wherein the first codon corresponds to position 48 using Kabat numbering and the second codon corresponds to position 64 using Kabat numbering. The invention further comprises use of the method of embodiment 19 for creation of an expression library. In one embodiment, the library is the library of embodiment 18.
(158) Embodiment 20 comprises the method of making a mutant composition comprising an immunoglobulin scaffold having one or more improvements selected from increased stability, increased melting temperature, increased solubility and decreased aggregation, wherein the method is the method of embodiment 19. The improvements of a mutant composition of Embodiment 20 may be selected from improved thermal refolding efficiency, increased resistance to protease digestion, increased shelf life at or above room temperature, at 4 C., or below 0 C. The improvements of a mutant composition of Embodiment 20 may be further characterized as increased or prolonged functionality when administered to a human subject when compared to a corresponding composition that does not comprise the non-canonical disulfide bond.
(159) Embodiment 21 comprises any one or more compositions of any one or more of embodiments 1 through 14, wherein the composition is a therapeutic agent. The invention further comprises the composition of embodiment 21 in a pharmaceutically acceptable carrier or excipient therefor.
(160) Embodiment 22 comprises the use of a composition of embodiment 21, wherein the composition is administered to a subject in need thereof. Embodiment 23 comprises the use of any one or more compositions of embodiment 21 in a medicament to be administered to a subject in need of treatment.
(161) Embodiment 24 comprises any one or more compositions of any one or more of embodiments 1 through 14, wherein the composition comprising the immunoglobulin scaffold is derived from a non-human animal. In one embodiment, the non-human animal includes all vertebrates, especially vertebrates selected from avians, amphibians, reptiles, mammals, camelids, chicken, rat, mouse, rabbit, goat, sheep, cow, cat, dog, horse, or nonhuman primates. In one embodiment the immunoglobulin scaffold is human. In one embodiment the immunoglobulin scaffold is camelid. In one embodiment, the camelid species is selected from the group consisting of llama, alpaca and camel. In one embodiment, the camelid scaffold is derived from any one or more of a V.sub.H region, a V.sub.L or a V.sub.HH region.
(162) TABLE-US-00013 SEQUENCES CloneName Sequence SEQIDNO HVHAm302 QVQLVESGGGLIKPGGSLRLSCAASGDTVSDESMTWVRQAPGKGLEW 1 VSAISSSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVY YCVTDNRSCQTSLCTSTTRSWGQGTMVTVSS HVHAm302S QVQLVESGGGLIKPGGSLRLSCAASGDTVSDESMTWVRQAPGKGLEW 2 V AISSSGGSTYYADSVKGRFT
SRDNSKNTVLYQMNSLRAEDTA VYYCVTDNRSCQTSLCTSTTRSWGQGTMVTVSS HVHAm427 QVQLVESGGGLIKPGGSLRLSCAASGVTLSPECMAWVRQAPGKGLEW 3 VSAISSSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVY YCVSCEGENAFWGQGTMVTVSS HVHAm427S QVQLVESGGGLIKPGGSLRLSCAASGVTLSPECMAWVRQAPGKLGEW 4; V
AISSSGGSTYYADSVKGRFT
SRDNSKNTVYLQMNSLRAEDTA VYYCVSCEGENAFWGQGTMVTVSS HVHAm431: QVQLVESGGGLIKPGGSLRLSCAASGYTVSSECMGWVRQAPGKGLEW 5 VSAISSSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVY YCVRDSKNCHDKDCTRPYCSWGQGTMVTVSS HVHAm431S QVQLVESGGGLIKPGGSLRLSCAASGYTVSSECMGWVRQAPGKGLEW 6 V
AISSSSGSTYYADSVKGRFT
SRDNSKNTVYLQMNSLRAEDTA VYYCVRDSKNCHDKDCTRPYCSWGQGTMVTVSS HVHPC235 QVQLVESGGGLIKPGGSLRLSCAASGFSVISESMTWVRQAPGKGLEW 7 VSAISSSGGSTYYADSVKGRFTISRDNSKNTVHLQMNSLRADDTAVY YCAAKKIDGARYDYWGQGTMVTVSS HVHPC235S QVQLVESGGGLIKPGGSLRLSCAASGFSVISESMTWVRQAPGKGLEW 8 V
AISSSGGSTYYADSVKGRFT
SRDNSKNTVHLQMNSLRAEDTA VYYCAAKKIDGARYDYWGQGTMVTVSS HVLP324 DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLL 9 IFAASTLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQQSYST PRTFGHGTKVTVL HVLP324S DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLL 10
FAASTLQSGVPSRFS
SGSGTDFTLTISNLQPEDFATYYCQQSY STPRTFGHGTKVTVL HVLP325 EIVLTQSPTTLSLSPGERATLSCRASQSVGRYLAWYQQRPGQAPRLL 11 VFDTSNRAPGVPARFSGRGSGTLFTLTISSLEPEDSAVYFCQQRSSG LTFGGGTKVTVL HVLP325S EIVLTQSPTTLSLSPGERATLSCRASQSVGRYLAWYQQRPGQAPRLL 12
FDTSNRAPGVPARFS
RGSGTLFTLTISSLEPEDSAVYFCQQRS SGLTFGGGTKVTVL HVLP335 EIVMTQSPATLSLSPGERATLSCRASQSVSSSSLAWYQQKPGQAPRL 13 LIYGTSNRATGIPDRFSGSGSGTHFTLTINRLEPGDFAVYYCQQYGS SPRTFGQGTKVEIK HVLP335S EIVMTQSPATLSLSPGERATLSCRASQSVSSSSLAWYQQKPGQAPRL 14 L
YGTSNRATGIPDRFS
SGSGTHFTLTINRLEPGDFAVYYCQQY GSSPRTFGQGTKVEIK HVLP342 DIQMTQSPSSLSASVGDRVTITCRASQDIRTDLDWFQQRPGRAPHRL 15 IYGASSLQGGVPSRFSGSGSGTEFTLTISGLQPEDFATYYCLQHHTY PRTFGLGTKVTVL HVLP342S DIQMTQSPSSLSASVGDRVTITCRASQDIRTDLDWFQQRPGRAPHRL 16
YGASSLQGGVPSRFS
SGSGTEFTLTISGLQPEDFATYYCLQHH TYPRTFGLGTKVTVL HVLP351 EIVMTQSPVTLSLSPGERATLSCRASQSVGTSLAWYQQKPGQAPRLL 17 IYDASNRATGISARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRYNW PRTFGGGTKVTVL HVLP351S EIVMTQSPVTLSLSPGERATLSCRASQSVGTSLAWYQQKPGQAPRLL 18
YDASNRATGISARFS
SGSGTDFTLTISSLEPEDFAVYYCQQRY NWPRTFGGGTKVTVL HVLP364 ETTLTQSPATLSVSPGERATFSCRASQSVSNNLAWYQQKPGQAPRLL 19 IYGASSRTTGIPDRFSASGSGTDFTLTISRLEPEDFAVYYCQQYDTS PRTFGQGTKVEIK HVLP364S ETTLTQSPATLSVSPGERATFSCRASQSVSNNLAWYQQKPGQAPRLL 20
YGASSRTTGIPDRFS
SGSGTDFTLTISRLEPEDFAVYYCQQYD TSPRTFGQGTKVEIK HVLP389 QSVVTQPPSVSAAPGQRVTISCSGSSYNIGENSVSWYQQLPGTAPKL 21 LIYGNDKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDS NLRASVFGGGTKVTVL HVLP389S QSVVTQPPSVSAAPGQRVTISCSGSSYNIGENSVSWYQQLPGTAPKL 22 L
YGNDKRPSGIPDRFS
SKSGTSATLGITGLQTGDEADYYCGTW DSNLRASVFGGGTKVTVL HVLP3103 ETTLTQSPGTLSLSPGERATLSCRASQSVRNNLAWYQQRPGQAPRLL 23 IYGASTRATGIPARFSGSGSGTDFTLTISSLQVEDVAVYYCQQYYTT PKTFGQGTKVEIK HVLP3103S ETTLTQSPGTLSLSPGERATLSCRASQSVRNNLAWYQQRPGQAPRLL 24
YGASTRATGIPARFS
SGSGTDFTLTISSLQVEDVAVYYCQQYY TTPKTFGQGTKVEIK None SCQTSLCTSTTR 25 LSCAASGDTVSDESMTWVR 26 AEDTAVYYCVTDNR 27 SCQTSLCTSTTR 28 GLEWVCAISSSGGSTYYADSVK 29 FTCSR 30 LSCAASGDTVSDESMTWVR 31 AEDTAVYYCVTDNR 32 GLEWVCAISSSGGSTYYADSVK 33 FTCSR 34 LSCAASGFSVISESMTWVR 35 AEDTAVYYCAAK 36 M13RPa 5-TCACACAGGAAACAGCTATGAC-3 37 KT131 5-ACCACTACTACTAATAGCGCAGACCCACTCCAGCCCCTTC-3 38 M13FP 5-CGCCAGGGTTTTCCCAGTCACGAC-3 39 K1129 5-GCAGACTCCGTGAAGGGCCGATTCACCTGCTCCAGAGACAAT 40 TCCAAGAAC-3 KT130 5-TGCGCTATTAGTAGTAGTGGTGGTAGCACATACTACGCAGAC 41 TCCGTGAAGGGCCG-3 VTITCR 42 LLCFASTLQSGVPSR 43 FSCSGSGTDFTLTISNQPEDFATYYCQQSYSTPR 44 ATLSCR 45 GSGTLFTLTISSLEPEDSAVYFCQQR 46 LLCFDTSNR 47 FSCR 48 LLCYGTSNR 49 FSCSGSGTHFTLTIVR 50 ATLSCR 51 LEPGDFAVYYCQQYGSSPR 52 VTITCR 53 LCYGASSLQGGVPSR 54 FSCSGSGTEFTLTISGLQPEDFATYYCLQHHTYPR 55 ATLSCR 56 LLCYDASNR 57 FSCSGSGTDFTLTISSLEPEDFAVYYCQQR 58 LLCYGASSRTR 59 FSCSGSGTDFTLTISR 60 ATFSCR 61 LEPEDFAVYYCQQYDTSPR 62 LLCYGNDK 63 FSCSK 64 VTISCSGSSYNIGENSVSWYQQLPGTAPK 65 SGTSATLGITGLQTGDEADYYCGTWDSNLR 66 ATLSCR 67 LLCYGASTR 68 FSCSGSGTDFTLTISSLQVEDVAVYYCQQYYTTPK 69 HVHP44S EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW 70 V
AISGSGGSTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAEDTA VYYCAKDEPRSVSGLRGVVDSWGRGTLVTVSS HVHB82S QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW 71 V
AISGSGGSTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAEDTA VYYCGTDMEVWGKGTTVTVSS HVHP421S QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW 72 V
AISGSGGSTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAEDTA VYYCAKDGKGGSSGYDHPDYWGQGTLVTVSS HVHP419S QLQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW 73 V
AISGSGGSTYYADSVKGRFT
SRDNSKNSLYLQMNSLGAEDTA VYYCARSWSGSSYGGDLDSWGQGTLVTVSS HVHP430S QVQLVESGGGLIKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEW 74 V
AISSSGGSTYYADSVKGRFT
SRDNSKNTVYLQMNSLRAEDTA VYYCVREEYRCSGTSCPGAFDIWGQGTMVTVSS HVHP429S EVQLVESGGTLVQPGGSLRLSCAASGFTFINYAMSWVRQAPDKGLDW 75 V
TISNNGGATYYADSVKGRFT
SRDNSNNTLYLQMNSLRPDDTA VYYCAKGPINTGRYGDWGQGTLVTVSS HVHM41S QVQLVQSGGGLVQPGRSLRLSCAASGFAFSSYAMSWVRQAPGKGLEW 76 V
AISGGGDHTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAEDTA VYYCAKEGMVRGVSSAPFDYWGQGTLVTVSS HVHM81S EVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEW 77 V
GISGSGASTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAGDTA LYYCARQSITGPTGAFDVWGQGTMVTVSS HVHP428S QLQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWFRQAPGKGLEW 78 V
FIRSKAYGGTTEYAASVKGRFT
SRDDSKSIAYLQMNSLRAED TAMYYCARRAKDGYNSPEDYWGQGTLVTVSS HVHP420S QVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMTWVRQAPGKGLEW 79 V
RIKTKTDGGTTDYAAPVKGRFT
SRDDSKNTLYLQMNSLKTED TAVYYCTTDRDHSSGSWGQGTLVTVSS HVHP414S DVQLVQSGGGLVKPGGSLRLSCTASGFPFSNAWMSWVRQAPGKGLEW 80 V
RITSKTDGGTTDYVAPVKGRFT
SRDDSKNTLYLQMNSLKTED TAVYYCTTDQANAFDIWGQGTMVTVSS HVHP423S QMQLVQSGGGVVQPGGSLRLSCAASGFTVSSSRMSWFRQAPGMGLEW 81 V
VIYSGGSTYYADSVRGRFS
SRDNSKNTLYLQMNSLRAEDTAL YYCAREREGAVTREDWGQGTLVTVSS HVHP413S QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEW 82 V
FIYSGGSTYYADSVKGRFT
SRDNSKNTLYLQMNSLRAEDTAV YYCARESRVGGGAFDIWGQGTMVTVSS HVHP426S QVQLVQSGGGVVQPGRSLRLSCAASGFIVDGYAMHWVRQAPGQGLEW 83 V
VTNNGGSTSYADSVKGRFT
SRDNSKNTVYLQMNSLRAEDTAV YYCARQSITGPTGAFDIWGQGTMVTVSS QAPGQGVEWVCVTNNGGSTSADSVK 84 FTCSR 85 GSGSKAAA 86 V.sub.L24-CDR1 AGAGTCACCATCACTTGCNNKGCAAGTCAGRGCATT 87 NNKNNKNNKTTANNKTGGTATCAGCAGAAACCA V.sub.L24S-CDR2 CCTAAACTCCTGTGCTTTNNKGCATCCNNKCKNNNK 88 AGTGGGGTCCCATCAAGG V.sub.L24-CDR3 TTTGCAACTTACTACTGTNNKCAGNNKNNKNNKNNK 89 CCTNNKACGTTCGGCCACGGGACC V.sub.L24-CDR3a TTTGCAACTTACTACTGTNNKCAGNNKNNKNNKNNK 90 CCTNNKNNKACGTTCGGCCACGGGACC V.sub.L24-CDR3b TTTGCAACTTACTACTGTNNKCAGNNKNNKNNKNNK 91 CCTNNKNNKNNKACGTTCGGCCACGGGACC 96GIII CCCTCATAGTTAGCGTAACGATCT 92 fdTGIII GTGAAAAAATTATTATTCGCAATTCCT 93 HV.sub.L24-BamHI TTGTTCGGATCCTAGGACGGTCACCT 94 HVL24-Bbs1 TATGAAGACACCAGGCCGACATCCAG 95 KT124 TGTGCTACCACCACTACTACTAATAGCGCAGACCCA 96 CTCCAGCCCCTTCCCTGG KT125 CACGGTGTTCTTGGAATTGTCTCTGGAGCAGGTGAA 97 TCGGCCCTTCACGGAGTC M13RPb CAGGAAACAGCTATGAC 98 HVLHLNN DIQMTQSPSSLSASVGDRVTITCSASQSINNRLYWYQQKPGKAPKLL 99
FPASFLFSGVPSRFS
SGSGTDFTLTISNLQPEDFATYYCQQSY STPRTFGHGTKVTVL HVLHLDS DIQMTQSPSSLSASVGDRVTITCTVSQGIDSRLYWYQQKPGKAPKLL 100
FPASLLESGVPSRFS
SGSGTDFILTISNLQPEDFATYYCQQSY STPRTFGHGTKVTVL HVLHLRN DIQMTQSPSSLSASVGDRVTITCKASQGIRNRLYWYQQKPGKAPKLL 101
FPASILDSGVPSRFS
SGSGTDFTLTISNLQPEDFATYYCAQQP GLPNSRTFGHGTKVTVL HVLHLNE DIQMTQSPSSLSASVGDRVTITCDASQSINERLYWYQQKPGKAPKLL 102
FPASILTSGVPSRFS
SGSGTDFTLTISNLQPEDFATYYCRQPR SGPSTFGHGTKVTVL HVLHLAQ DIQMTQSPSSLSASVGDRVTITCGASQSIAQRLYWYQQKPGKAPKLL 103
FPASLLHSGVPSRFS
SGSGTDFTLTISNLQPEDFATYYCAQRA SPPRPTFGHGTKVTVL HVLHLEM DIQMTQSPSSLSASVGDRVTITCQASQGIEMFLQWYQQKPGKAPKLL 104
FAASTLQSGVPSRFS
SGSGTDFTLTISNLQPEDFATYYCVQPG VAPPPGTFGHGTKVTVL HVLHLQI DIQMTQSPSSLSASVGDRVTITCRASQSIQIMLDWYQQKPGKAPKLL 105
FGASFLISGVPSRFS
SGSGTDFTLTISNLQPEDFATYYCRQTW TPPAPTFGHGTKVTVL HVLHLYS DIQMTQSPSSLSASVGDRVTITCQASQSIYSKLYWYQQKPGKAPKLL 106
FPASLLWSGVPSRFS
SGSGTDFTLTISNLQPEDFATYYCLQNA ADPHRTFGHGTKVRL HVLHSA1 DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLL 107
FGASRLNSGVPSRFS
SGSGTDFTLTISNLQPEDFATYYCRQLF PLPDRRTFGHGTKVTVL HVLHLFL DIQMTQSPSSLSASVGDRVTITCLASQSIFLYLDWYQQKPGKAPKLL 108
FFASTLESGVPSRFS
SGSGTDFTLTISNLQPEDFATYYCQQSY STPRTFGHGTKVTVL
(163) The embodiments and examples described herein are illustrative and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Furthermore, the discussed combination of features might not be necessary for the inventive solution.
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