Patent Application
20250051478
The present invention relates to variants of scFv and antibodies having a reduced tendency of forming multimers. In particular the invention relates to bispecific antibodies comprising said scFvs.
The invention further relates to pharmaceutical compositions comprising one or more antibodies of the invention and the use thereof for treatment of cancer.
scFv fragments and antibodies, such as bispecific antibodies, have found wide use in antibody assisted diagnosis and therapy.
Pretargeted radioimmunotherapy (PRIT) is one example of a pharmaceutical method that has utilized the efficient binding of antibodies to a specific target allowing treatment to be focused at the targeted site.
WO2018204873 discloses the SADA technology, which benefits from SADA domains having the capability of assembling or disassembling dependent on concentration. This property is particularly beneficial in connection with PRIT. A bispecific antibody capable of binding a cytotoxic agent and a target site, connected to a SADA domain can be administered in multimeric form, in particular tetrameric form, and bind to the antibody target site, whereas unbound molecules will disassemble and be removed from the plasma stream via the kidneys before the cytotoxic agent is administered.
It is important for SADA-PRIT therapies that the molecular size of the multimeric form, in particular tetrameric form, is above the renal clearance limit and thereby providing a long plasma half-life, and that the monomeric form is below the renal clearance limit, providing a short plasma half-life.
For pharmaceutical compositions comprising one or more antibodies it is also important that the one or more antibodies remains stable during the shelf life of the pharmaceutical composition, avoiding degradation of the antibodies as well as unintended agglomeration or cross reactions between antibodies and/or between antibodies and other components of the composition.
In a first aspect the invention relates to a method of generating variants of a scFv domain, comprising a light chain variable domain (VL), a heavy chain variable domain (VH) and one or more disulfide bonds between the VL and the VH, comprising the steps of
The variant scFvs of the invention, which are scFvs by themselves, have reduced ability and/or tendency to form multimers compared with scFvs with a disulfide bond between the VH and VL domain.
In another aspect the invention relates to scFv domains prepared according to the method of the invention.
In a further aspect the invention relates to bispecific antibodies comprising a first scFv domain capable of binding DOTA or DOTAM metal chelate, a second scFv domain capable of binding a tumor antigen, and a SADA domain, wherein the first scFv domain and/or the second scFv domain do not contain a disulfide bond between the VH and VL domains.
In a further aspect the invention relates to compositions, in particular pharmaceutical compositions, comprising a scFv or bispecific antibody of the invention, and the use of such compositions for diagnosing or treating cancer.
In a further aspect the invention relates to a kit comprising a scFv or bispecific antibody of the invention.
The invention also relates to polynucleotides, expression vectors or constructs, comprising such polynucleotides, host cell comprising such a polynucleotide or expression vector or constructs and the use of such host cells for the preparation of the scFv or bispecific antibodies on the invention.
Additional aspects are provided in the claims.
Some embodiments of the present invention are provided in the claims.
In one embodiment the invention relates to a method of generating variants of a scFv domain, comprising a light chain variable domain (VL), a heavy chain variable domain (VH) and one or more disulfide bonds between the VL and the VH, comprising the steps of
According to the invention one or all of the disulfide bonds identified in step a., may be removed by substituting the cysteines forming said bond(s) with other amino acids. It is preferred to substitute both cysteines forming a disulfide bond with other amino acids in order to avoid any free cysteine.
The invention is based on the observation that many scFv domains and constructs comprising scFv domains may form multimers, such as dimers or trimers; or multiple forms of the monomer form. This is not desirable for compounds intended for pharmaceutical use, where high uniformity and purity of the compounds are generally desired. Further, heterogenicity of scFv domains and constructs comprising scFv domains complicate recovery and purification compared with similar compounds having a higher homogenicity.
The inventors have realized that disulfide bonds between the VH and VL of the scFv are responsible for multimerization and formation of alternative disulfide bonding leading to the observed formation of multimers and multiple forms of the scFvs, and that scFvs without disulfide bonds between the VH and VL domains have less tendency of forming multimers or multiple forms can be provided using the method of the invention.
The obtained scFvs have similar binding properties as the scFvs from which the variants were derived according to the method of the invention. The skilled person will further realize that the variants derived from a scFv according to the invention are in fact also a scFv in itself.
In natural antibodies the VL and VH sequences are part of the Light and Heavy immunoglobulin chains and in nature the light and heavy chains are connected by one or more disulfide bonds found in the constant regions adjacent to the VL and VH sequences. However, since a scFv consists of only the VL and VH sequences connected by a linker, the disulfide bonds, found in the constant regions adjacent to the VL and VH sequences, and which in natural antibodies connects the chains containing the VL and VH chains, are not present in scFvs and it is therefore common practice to introduce a disulfide bond in scFvs, between the VL and VH sequences in order to improve stability of the scFv. The invention is based on the inventor's realization that such an introduced stabilizing disulfide bonds between the VH and VL domain of an scFv may lead to heterogenicity, which may be disadvantageous for at least some uses of the scFv.
It is known in the area that VH and VL domains may comprise additional disulfide bonds between two cysteine residues in the same domain (intradomain disulfide bonds), and the inventors have further realized that these intradomain disulfide bonds, in contrast to interdomain disulfide bonds (between the VL domain and the VH domain) are not important, or at least less important, for the observed heterogenicity.
In one embodiment the invention relates to a method of generating variants of a scFv domain, wherein said variants give rise to less multimer formation compared with the original scFv domain.
In another embodiment the invention relates to the use of an scFv without a disulfide bond between the VH and VL domain in a polypeptide construct comprising the scFv and an additional domain, where the polypeptide construct has low tendency of multimerize or at least less tendency of forming multimers compared with a similar polypeptide having a disulfide bond between the VH and VL domains of the scFv that, except for this additional disulfide bond, has same sequence.
Multimer formation may be detected using techniques known in the art for determining molecular weights for example chromatographic methods.
In one embodiment of the invention, multimer formation is determined by SDS-PAGE gelelectrophoresis.
In one embodiment of the invention the scFv domain is part of a polypeptide comprising additional antibody fragments. For example, the scFv may be part of a polypeptide that in addition to the scFv domain comprises one or more of: additional scFv domains, immunoglobulin heavy and/or light chains, Fc domains, hinge regions etc.
In one preferred embodiment, the scFv domain is part of a bi- or a multispecific antibody.
One form of such a bi- or multispecific antibody is a bispecific antibody comprising two immunoglobulin heavy chains and two chains of a fusion polypeptide comprising an immunoglobulin light chain C-terminally fused to an scFv domain, where a first binding specificity is provided by the variable regions of the immunoglobulin heavy and light chains and a second binding specificity is provided by the scFv domains.
Another form of such a bi- or multispecific antibody is a polypeptide comprising two or more scFv domains, each providing a binding specificity.
In one embodiment, the invention relates to a bi- or multispecific antibody further comprising a SADA domain, also known as a tetramerization domain.
SADA (self assembly and disassembly) domains are short amino acid domains capable of spontaneously assembling and disassembling in solution, depending on concentration. Complexes comprising a SADA domain typically exists in at least two distinct forms, a tetrameric form at high concentration and a monomeric form at low concentration. The self assembly and disassembly (SADA) technology has been disclosed in WO 2018204873A1, which is incorporated in its entirety by reference.
SADA-complexes may be designed so that the tetrameric form has a molecular weight well above the renal clearance limit and the monomeric form has a molecular weight below the renal clearance limit, meaning that the tetrameric form will have a high plasma-half-life, because it is not excreted into the urine, and the monomeric form has a low plasma half-life, because it is excreted into the urine.
Preferred SADA domains for use according to the invention includes domains comprising the sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97% sequence identity to one of these sequences.
Preferred SADA domains for use according to the invention include domains having the sequence of:
Preferred SADA domains according to the invention are domains comprising a sequence with at least 80% sequence identity to amino acids 6-36 of SEQ ID NO: 5, and which differs from the sequence of SEQ ID NO:5 with one or more substitutions, wherein the domain maintains the ability to dimerize or tetramerize.
The skilled person can easily determine whether such a domain with a given substitution maintains the ability to dimerize or tetramerize by simple routine experimentation, or find such information in the literature, e.g. in J. Gencel-Augusto and G-Lozano; Genes & Development 34:1128-1146, incorporated by reference.
A preferred SADA domain according to the invention is a domain with an amino acid sequence that differs from the sequence of amino acids 6-36 of SEQ ID NO: 5 by 1, 2, 3, 4 or 5 substitutions selected among following substitutions:
The L24P substitution abolished tetramerization completely and should not be applied.
The p53 tetramerization domain comprising the sequence of amino acids 6-36 of SEQ ID NO: 5, is a preferred SADA domain.
The present invention is particular useful in connection with the SADA technology, using a construct comprising one or more scFvs and a SADA domain constructed so a tetrameric complex comprising four such monomers have a size above the renal clearance limit whereas the monomers of such complex have a size below the renal clearance limit. Typically, such a construct comprising one or more scFvs and a SADA domain has a molecular weight of about 50-60 kD in the monomeric form. The complex will after administration in its tetrameric form bind to its target and unbound complexes will disassemble in plasma and be rapidly excreted because its size is below the renal clearance limit. However, if part of the polypeptides form multimers mediated by disulfide bonds between the VH and VL of the scFv, theoretically and depending on the actual construct, the clearance of disassembled complexes may be less efficient because of the formed multimers.
It is therefore particular beneficial to use the present invention in connecting with the SADA technology to reduce the formation of multimers.
In one embodiment the invention relates to a scFv domain comprising a VL and a VH and capable of binding an antigen, wherein the scFv is obtainable according to the method of any of the preceding claims. Preferably, the VH and VL are not connected by any disulfide bond.
In one embodiment the scFv domain of the invention further comprises a linker between the VH and VL. Linkers also sometimes known as spacers are short amino acid sequences created to separate multiple domains in a single protein. Linkers are known in the art and the present invention is not limited to any particular sequence of the linkers. In general, the purpose of linkers is to connect and/or separate different elements of the complex and are typically mainly composed of small hydrophilic amino acids such as glycine, serin and threonine.
In one embodiment the invention relates to an antibody or antibody fragment capable of binding DOTA metal chelate, and comprises:
The antibodies and antibody fragments capable of binding DOTA may be derived from the murine antibody 2D12.5 that was affinity matured to increase the affinity to DOTA (WO 2010/099536). When a scFv was generated based on 2D12.5, a stabilizing disulfide bond was generated by inserting cysteines in positions corresponding to position 111 and 179 of SEQ ID NO: 3. This disulfide stabilized scFv was affinity matured in several rounds generating a number of modified antibodies having improved affinity for DOTA-metal, including the scFv named C825 (SEQ ID NO: 3). The inserted disulfide bond appears to have been considered essential because WO 2010/099526 explains that all variants that emerged from the last affinity maturation were discarded because they had lost the stabilizing disulfide bond.
C825 has later been frequently used, and it has been humanized in order to obtain an antibody having excellent DOTA-metal binding properties, giving rise to fewer adverse reactions when administered to humans, and it appears that the disulfide bond located between position 111 and 179 in the corresponding mC825 scFv SEQ ID NO: 3 invariable have been maintained.
Now the present inventors have surprisingly discovered that the disulfide bond between position 111 and 179 is not mandatory to obtain a functional antibody, in fact it is advantageous to remove this disulfide bond including the cysteines corresponding to positions 111 and 179 of mC825 scFv (SEQ ID NO 3).
The fact that the scFv is capable of binding DOTA metal chelate is intended to mean that the scFv is capable of specifically binding DOTA metal chelate, in particular DOTA binding 175Lu, with a binding constant Kd of about 10−4 M or less, e.g., in the range of 10−4M to 10−12 M, e.g., in the range of 10−5M to 10−10 M, e.g., in the range of 10−6M to 10−9 M.
In one embodiment, the scFv of the invention comprises VL and VH domains consisting of SEQ ID NO: 1 and 2.
In one embodiment, the scFv comprising or consisting of the sequence of SEQ ID NO: 4, or comprising or consisting of a sequence having at least 90% sequence identity, e.g. at least 95% sequence identity, e.g. at least 96% sequence identity, e.g. at least 97% sequence identity, e.g. at least 98% sequence identity or at least 99% sequence identity to SEQ ID NO: 4.
In one embodiment the scFv domain of the invention, is capable of binding GD2, and comprises:
The fact that the scFv is capable of binding GD2 is intended to mean that the scFv is capable of specifically binding GD2 with a binding constant Kd of about 10−4 M or less, e.g., in the range of 10−4M to 10−12 M, e.g., in the range of 10−5M to 10−10 M, e.g., in the range of 10−6M to 10−9 M.
In one embodiment, the scFv domain of the invention comprises or consists of the sequence of SEQ ID NO: 21, or comprises or consists of a sequence having at least 90% sequence identity, e.g. at least 95% sequence identity, e.g. at least 96% sequence identity, e.g. at least 97% sequence identity, e.g. at least 98% sequence identity or at least 99% sequence identity to SEQ ID NO: 21.
In one embodiment the scFv domain of the invention, is capable of binding CD38, and comprises:
The fact that the scFv is capable of binding CD38 is intended to mean that the scFv is capable of specifically binding CD38 with a binding constant Kd of about 10−4 M or less, e.g., in the range of 10−4M to 10−12 M, e.g., in the range of 10−5M to 10−10 M, e.g., in the range of 10−6M to 10−9 M.
In one embodiment the CDR sequences consists of SEQ ID NO: 22-27.
In one embodiment, the scFv of the invention, comprises or consists of the sequence of SEQ ID NO: 30, or comprises or consists of a sequence having at least 90% sequence identity, e.g. at least 95% sequence identity, e.g. at least 96% sequence identity, e.g. at least 97% sequence identity, e.g. at least 98% sequence identity or at least 99% sequence identity to SEQ ID NO: 30.
In one embodiment the scFv domain of the invention, is capable of binding CD20, and comprises:
The fact that the scFv is capable of binding CD20 is intended to mean that the scFv is capable of specifically binding CD20 with a binding constant Kd of about 10−4 M or less, e.g., in the range of 10−4M to 10−12 M, e.g., in the range of 10−5M to 10−10 M, e.g., in the range of 10−6M to 10−9 M.
Preferably, the CDR sequences consists of SEQ ID NO: 31-36.
In one embodiment, the scFv of the invention comprises or consists of the sequence of SEQ ID NO: 39, or comprises or consists of a sequence having at least 90% sequence identity, e.g., at least 95% sequence identity, e.g., at least 96% sequence identity, e.g., at least 97% sequence identity, e.g., at least 98% sequence identity or at least 99% sequence identity to SEQ ID NO: 39.
In one embodiment the scFv domain of the invention is capable of binding GPA33, and comprises:
The scFv of this embodiment may comprise or consist of the sequence of SEQ ID NO: 61, or of a sequence having at least 90% sequence identity, e.g. at least 95% sequence identity, e.g. at least 96% sequence identity, e.g. at least 97% sequence identity, e.g. at least 98% sequence identity or at least 99% sequence identity to SEQ ID NO: 61.
In one embodiment the scFv of the invention is capable of binding RSV and comprises
In one embodiment the scFv of the invention is capable of binding B7H3 and comprises
In one embodiment the scFv of the invention is capable of binding HER2 and comprises
In one embodiment the scFv of the invention is capable of binding HER2 and comprises
In one embodiment the scFv of the invention is capable of binding DOTAM and comprises
In one embodiment the invention relates to bispecific antibodies comprising a first and a second binding site, wherein at least one of the first and the second binding sites is a scFv that does not comprise a disulfide bond between the VH and VL domains.
Several forms for bispecific antibodies are known in the art and the present invention is not limited to any particular such form. One example of a bispecific antibody is an antibody comprising two antibody heavy chains and two fusion polypeptides, the fusion polypeptides comprising an antibody light chain where a scFv sequence is fused to the C-terminal of the light chain. Another example of a bispecific antibody is a molecule comprising two or more scFv sequences linked serially after each other.
In one embodiment, the bispecific antibody of the invention comprises a first scFv domain that does not comprise a disulfide bond connecting the VH and the VL and/or a second scFv domain that does not comprise a disulfide bond connecting the VH and the VL.
In one embodiment, the bispecific antibody of the invention further comprises one or more linker sequences.
In one preferred embodiment the invention relates to a bispecific antibody comprising a first scFv domain capable of binding a chelator, a second scFv domain capable of binding a tumor antigen, and a SADA domain, wherein the first scFv domain and/or the second scFv domain do/does not comprise a disulfide bond between the VH and VL domains. Preferably, the first and or the second scFv is a scFv of the invention. The tumor antigen may be any antigen known to be present mainly on the surface of tumor cells, in particular on the surface of solid tumors.
Examples of such tumor antigens include: HER2, B7-H3, CA6, CD138, CD20, CD19, CD22, CD27L, CD30, CD33, CD37, CD38, CD47, CD56, CD66e, CD70, CD74, CD79b, EGFR, EGFRvIII, FRα, GCC, GPNMB, Mesothelin, MUC16, NaPi2b, Nectin 4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, alpha v beta6, CA19.9, CAIX, CD138, CD174, CD180, CD227, CD326, CD79a, CEACAM5, CRIPTO, DLL3, DS6, Endothelin B receptor, FAP, GD2, Mesothelin, PMEL 17, SLC44A4, TENB2, TIM-1, CD98, Endosialin/CD248/TEM1, Fibronectin Extra-domain B, LIV-1, Mucin 1, p-cadherin, peritosin, Fyn, SLTRK6, Tenascin c, VEGFR2, and PRLR.
Preferred examples of tumor antigens include GD2, CD38, B7-H3, CD33, GPA33.
GD2 is a disialoganglioside expressed on tumors of neuroectodermal origin, such as neuroblastoma and melanoma. The expression on normal tissue is highly restricted.
CD38, also known as cyclic ADP ribose hydrolase, is found on the surface of many immune cells including CD4+, CD8+, B lymphocytes and natural killer cells. The expression is very high on myeloma cells.
B7-H3, B7 homolog 3, also known as CD276 is a type I transmembrane protein that exists in two isoforms. It has limited expression in normal tissue and is expressed at high frequency on many different cancer types e.g. neuroblastoma.
CD33, also known as sialic acid binding Ig-like lectin 3, is a cell surface antigen. It is expressed on cells of myeloid lineage. It can be aberrantly expressed on some cases of plasma-cell myeloma.
GPA33, Glycoprotein 33, is a cell surface antigen that is expressed in greater than 95% of human colon cancers.
The binding site capable of binding a chelator, or a chelator binding a metal ion, may be any such binding site known in the art. Preferred examples of chelators include DOTA, DOTAM, and variants of these. Examples of suitable binding sites capable of binding a chelator or a chelator binding a metal ion may be found in WO 2010/099539, disclosing binding sites capable of binding DOTA or derivatives of DOTA and which binding sites based on the antibody 2D12.5, and WO 2019/201959, disclosing rabbit antibodies capable of binding DOTAM, incorporated by reference.
In one embodiment, the bispecific antibody of the invention further comprises a SADA domain
Preferably the bispecific antibody of the invention comprises:
In one example, the bispecific antibody of the invention comprises:
In another example, the bispecific antibody of the invention comprises:
Preferred examples of bispecific antibodies of the invention include the bispecific antibodies comprising or consisting of one of the sequences SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69.
In one embodiment, the invention relates to a composition comprising a scFv or a bispecific antibody of the invention. Preferably, the composition is a pharmaceutical composition.
In one embodiment the invention relates to the use of a bispecific antibody of the invention, for diagnosing or treating cancer.
The cancer is preferably a solid cancer or tumor.
In one embodiment the invention relates to the use of a bispecific antibody according to the invention in a method comprising the steps:
In one embodiment the invention relates to the use of a bispecific antibody comprising a first scFv capable of binding DOTA-metal or DOTAM-metal, a second scFv capable of binding a tumor antigen and a SADA-domain, according to the invention in a method comprising the steps:
In this embodiment the bispecific antibody comprising a first scFv capable of binding DOTA-metal, DOTAM-metal or a derivative thereof, a second scFv capable of binding a tumor antigen and a SADA-domain is preferably administered in a tetrameric form.
The holding period should be selected in order to give sufficient time to allow the bispecific antibody to find and bind to tumor antigen and to allow the unbound bispecific antibody in tetrameric form to disassemble into monomeric form and thereby quickly be cleared from the blood stream.
The holding period may be selected in the range of 48-96 hours.
In one embodiment the method further comprises comprising administering a clearing agent after step a and before step b.
In one embodiment of the invention, DOTA or derivative thereof is selected among DOTA, Benzyl DOTA and the bischelate compound
In one embodiment the radionuclide is selected from the group consisting of 211At, 51Cr, 57Co, 58Co, 67Cu, 152Eu, 67Ga, 111In, 59Fe, 212Pb, 177Lu, 223Ra, 224Ra, 186Re, 188Re, 75Se, 99mTc 227Th, 89Zr, 90Y, 94mTc, 64Cu, 68Ga, 66Ga, 86Y, 82Rb, 110mIn, 209Bi, 211Bi, 212Bi, 213Bi, 210Po, 211Po, 212Po, 214Po, 215Po, 216Po, 218Po, 211At, 215At, 217At, 218At, 221Fr, 223Ra, 224Ra, 226Ra, 225Ac, 227Ac, 227Th, 228Th, 229Th, 230Th, 232Th, 231Pa, 233U, 234U, 235U, 236U, 238U, 237Np, 238Pu, 239Pu, 240Pu, 244Pu, 241Am, 244Cm, 245Cm, 248Cm, 249Cf, and 252Cf, preferable among 177Lu, 99mTc, 64Cu and 89Zr.
The chelator binding a radionuclide, such as DOTA or DOTAM or any derivative thereof bound to a radionuclide; may be administered twice or even more times. When the chelator binding a radionuclide is administered more than once it is recommended that the individual administrations are separated by 24 hours or more.
The two or more administrations of a chelator binding a radionuclide may be using the same radionuclide or, it may be using different radionuclides for each administration.
Such repeated administration of a chelator binding a radionuclide, such as DOTA or a derivative thereof binding a radionuclide; has been disclosed in WO 2021/242848 (incorporated by reference), with respect to GD2-SADA, however, the present inventors have realized that methods using repeated administration of a chelator, such as DOTA; binding a radionuclide is not necessarily limited to GD2-SADA, but can be applied to the bispecific antibodies of the invention.
In one example, a bispecific antibody of the invention, capable of binding a tumor antigen, is administered to a patient in need of such treatment or diagnosis; after 48 hours a chelator binding an alpha-emitter is administered to the patient, 24 hours after the administration of chelator binding the alpha-emitter a second administration of chelator binding a beta-emitter is administered. By using such a method, the benefit of treating using an alpha-emitter and the benefits of a beta-emitter is combined.
In another example, a bispecific antibody of the invention, capable of binding a tumor antigen, is administered to a patient in need of such treatment or diagnosis; after 48 hours a chelator binding a radionuclide suitable for PET or SPECT scanning is administered to the patient and a PET or SPECT scanning is performed. Depending on the outcome of the scanning a treatment procedure can be initiated by administering a chelator binding a radionuclide suitable for treating cancer 24 hours after the first administration of chelator binding a radionuclide, and the treatment may even be continued by administrating a second or subsequent dose of a radionuclide suitable for treating cancer.
Thus, one embodiment the invention relates to the use of a bispecific antibody according to the invention in a method comprising the steps:
In one embodiment the method further comprises detecting the localization of the radionuclide. The detection may be performed using well known methods and equipment for detecting radionuclides, such as a PET or SPECT scanner.
In one embodiment the cancer is selected among osteosarcoma, liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, spindle cell sarcoma, brain tumor, small cell lung cancer, retinoblastoma, HTLV-1 infected T cell leukemia.
The invention further relates to a kit comprising a bispecific antibody according to the invention. Preferably, the kit further comprises chelator, such as DOTA, DOTAM or a derivative of DOTA.
The kit may further comprise instructions for use or a link to such instructions.
The scFv domains and/or the bispecific antibodies of the invention may be prepared using methods known in the art.
One preferred method of providing the scFv domains and/or bispecific antibodies of the invention is to provide a polynucleotide encoding the desired scFv or bispecific antibody, providing the polynucleotide with suitable regulatory sequences, such as promoter, terminator, enhancer, ribosome binding site, Kozak sequence, polyadenylation site etc; inserting the construct in a suitable host cell that subsequently is grown under conditions leading to expression of the desired scFv or bispecific antibody.
The polynucleotide sequence may be assembled using techniques known in art e.g. using PCT technologies or is may be synthesized, for examples using commercial provides for such sequences.
Thus, in one embodiment the invention relates to a polynucleotide encoding a scFv domain or a bispecific antibody of the invention; an expression vector or construct comprising such a polynucleotide sequence, or a host cell comprising the polynucleotide, expression vector or construct.
In one embodiment the invention relates to a method of producing a scFv or a bispecific antibody of the invention, comprising the steps of:
All cited references are incorporated by reference.
The accompanying Figures and Examples are provided to explain rather than limit the present invention. It will be clear to the person skilled in the art that aspects, embodiments, claims and any items of the present invention may be combined.
Unless otherwise mentioned, all percentages are in weight/weight. Unless otherwise mentioned, all measurements are conducted under standard conditions (ambient temperature and pressure). Unless otherwise mentioned, test conditions are according to European Pharmacopoeia 8.0.
The nucleotide sequence for the intended protein, including regulatory sequences for directing expression was synthesized and inserted into an expression vector.
The expression vector was transfected into CHO cells and transformants were grown in standard medium for expression of the proteins, whereafter the proteins were recovered from the broth.
Pre-cast gels, Thermo Fisher Bolt Bis-Tris 4-12% gels, were provided from Thermo Fisher Scientific, MA USA, and used according to the manufacturer's instructions. After electrophoresis gels were stained with Coomassie using the manufacturer's instructions.
The GD2-SADA construct with the amino acid sequence SEQ ID NO: 43 was prepared and purified.
The GD2-SADA construct comprises a GD2 scFv (amino acid no: 1-252), a DOTA binding scFv (amino acids 275-533) and a SADA domain (amino acids 545-583). The construct comprises a disulfide bond between the VH and VL of the GD2 scFv, formed by the cysteines C97 and C179, and one disulfide bond between the VH and VL of the DOTA binding scFv, formed by the cysteines C369 and C513.
The purified construct was analyzed using SE-HPLC (see
In order to resolve the inhomogeneity, a truncated version of the GD2-SADA, called GD2-SADA minus P53 domain, was prepared where the molecule was truncated after amino acid G533, meaning that the SADA domain was lost.
The truncated form was also analyzed by SE-HPLC, see
The experiments showed that the GD2-SADA construct formed multimers, mainly dimers, and that the multimerization was not alone caused by the SADA domain.
The GD2-SADA construct and the truncated form, prepared in Example 1, was further analyzed by SDS-PAGE chromatography, see
GD2-SADA and truncated GD2-SADA were separated under non-reducing conditions and consistently showed the presence of multimers, in particular dimers and trimers. When the truncated form was analyzed under reducing conditions all forms collapsed into the monomeric form, confirming that the observed multimerization was caused by disulfide bonds.
In this example variants of bispecific antibodies capable of binding CD20 and DOTA was generated. The anti-CD20 site was varied in the order of VH and VL regions and with or without a disulfide bond connecting VH and VL. The DOTA binding site was the scFv disclosed in SEQ ID NO: 4 and the SADA domain was the domain disclosed in SEQ ID NO: 5.
The amino acid sequences of the VL sequence of the anti-CD20 scFv is disclosed in SEQ ID NO: 37, and for forming the anti-CD20 scFv without disulfide bond the cysteine in position 99 was substituted with a Glycine (G). The amino acid sequence of the VH sequence of the anti-CD20 scFv is disclosed in SEQ ID NO: 38, and for forming the anti-CD20 scFv without disulfide bond the cysteine in position 44 was substituted with a Glycine (G).
The sequence of the construct Ri-3A is disclosed in SEQ ID NO: 39.
Following constructs were generated
The four constructs were separated on SDS-PAGE under reducing and non-reducing conditions (See
The figure shows that under non-reducing conditions, the constructs comprising a disulfide bond between VH and VH (Ri-2A and Ri-4A) formed high molecular weight multimers, and that the multimers content was strongly reduced or even absent in the constructs without a disulfide bond between VH and VH (Ri-1A and Ri-3A).
Under reducing conditions all four constructs collapsed into the monomeric form.
In this example variants of bispecific antibodies capable of binding CD38 and DOTA was generated. The DOTA binding site was based on the scFv disclosed in SEQ ID NO: 3 and is the scFv disclosed in SEQ ID NO: 4. The DOTA binding site comprising one disulfide bond between the VH and VL containing cysteines in positions 111 and 194. The SADA domain was the domain disclosed in SEQ ID NO: 5.
The amino acid sequence of the VL sequence of the anti-CD38 scFv is disclosed in SEQ ID NO: 28, and for forming the anti-CD38 scFv without disulfide bond the cysteine in position 100 was substituted with a Glutamine (Q). The amino acid sequence of the VH sequence is disclosed in SEQ ID NO: 29, and for forming the anti-CD38 scFv without disulfide bond the cysteine in position 44 was substituted with a Glycine (G).
Following constructs were generated
The constructs were analyzed on non-reducing SDS-PAGE, See
The results show that YMS9a and YMS9c contained significant amounts of multimers, whereas the amount of multimers were significantly diminished or absent in YMS9d.
The results also showed that YMS9a and YMS9c gave rise to some heterogeneity in the monomer band. The heterogeneity disappeared under reducing conditions.
The YMS9d product was analysed further by loading various amounts, 3.2 μg, 1.6 μg, 1.1 μg and 0.5 μg on SDS-PAGE gel under non-reducing and reducing conditions. The results, shown in
In this example, the binding properties of a SADA construct of the invention were tested by SPR analysis.
The YMS9a (with a disulfide bond between the VL and VH of the DOTA binding site and a disulfide bond between the VL and VH of the CD38 binding site) and YMS9d (without disulfide bonds between VL and VH), prepared according to example 4, were analysed by SPR analysis both for binding to DOTA and for binding to CD38.
The results showed no significant difference for in vitro binding efficacy between YMS9a and YMS9d.
The compound YMS9d, as prepared in example 4, and a solution of 10 mg/ml was prepared. After the solution was prepared, it was allowed to equilibrate for 3 hours at room temperature. The solution was analysed by SE-HPLC.
A sample of the stock solution was upconcentrated to 20 mg/ml. After the solution was prepared, it was allowed to equilibrate for 3 hours at room temperature. The solution was analysed by SE-HPLC.
The 10 mg/ml solution and the 20 mg/ml solutions were each diluted to 1 mg/ml. After the solution was prepared, it was allowed to equilibrate for 3 hours at room temperature. The solutions were analysed by SE-HPLC.
The results are shown in
The binding properties of samples diluted from 10 mg/ml and 20 mg/ml solutions as described in example 6, were analysed by SPR. Results are shown in the tables below:
The results showed that the formation and subsequent disassembly of HMW forms did not significantly change the binding properties.
The compounds YMS9c, comprising one disulfide bond between the VH and VL chain in the CD38 scFv, and YMS9d, without any disulfide bonds between the VH and VL, were used in this example. The compounds were prepared as described in Example 4.
The compounds were labelled with 125I and incubated with Daudi cells, comprising the CD38 antigen exposed on their surface. After the incubation the cells were rinsed and the radioactivity bound to the cells counted:
The results showed that YMS9d, without VH-VL disulfides; had higher binding compared with YMS9c, with a disulfide bond on anti-CD38 site.
Daudi tumor bearing mice were given injections of 10 mg/kg of YMS9c, comprising one disulfide bond between the VH and VL chain in the CD38 scFv, and YMS9d, without any disulfide bonds between the VH and VL, as prepared in example 4.
48 h after administration of the CD38-SADA compounds, 5 MBq 177Lu-DOTA/177Lu-Bn-DOTA was administered to the mice.
2 hours and 24 hours after administration of radioactivity, the biodistribution was determined by euthanising and dissecting some mice (n=4) and counting the amount of radioactivity found in the selected tissues: Blood, tumor and kidney.
The tumor:blood ratios were calculated:
The example showed higher tumor:blood uptake of the CD38-SADA Conjugate without disulfide bond between VL and VH compared with the conjugate with a disulfide bond between the VH and VL of the CD38 scFv.
In this example variants of bispecific antibodies capable of binding RSV and DOTA was generated. The DOTA binding site was the scFv disclosed in SEQ ID NO: 4 and the SADA domain was the domain disclosed in SEQ ID NO: 5.
Two versions of the RSV-SADA conjugate were generated, one version, PalDOT-SAD with a disulfide bond between the VL and VH of the RSV binding scFv, and one version, PA-3A without disulfide bonds between the VL and VH of the RSV binding scFv.
The sequence of the construct PA-3A is disclosed in SEQ ID NO: 62.
The two constructs were expressed and run on a non-reducing SDS-PAGE gel, as shown in
In this example variants of bispecific antibodies capable of binding B7H3 and DOTA was generated. The DOTA binding site was the scFv disclosed in SEQ ID NO: 4 and the SADA domain was the domain disclosed in SEQ ID NO: 5.
Two versions of the B7H3-SADA conjugate were generated, one version, 3BH-4 with a disulfide bond between the VL and VH of the B7H3 binding scFv, and one version, 3BH-5 without disulfide bonds between the VL and VH of the B7H3 binding scFv.
The sequence of the construct 3BH-5 is disclosed in SEQ ID NO: 63.
The two constructs were expressed and run on a non-reducing SDS-PAGE gel, as shown in
In this example variants of bispecific antibodies capable of binding HER2 and DOTA were generated. The SADA domain was the domain disclosed in SEQ ID NO: 5.
Eight versions of the HER2-SADA conjugate were generated, one series (TR-series), using an anti-HER2 scFv derived from the clinical antibody, Trastuzumab, with four constructs:
And one series (PE-series), using an antiHER2 scFv derived from the clinical antibody; Pertuzumab, with four constructs:
The constructs of the TR series differ from the constructs of the PE series in that the VH and VL sequences of the anti-HER2 scFv site in the TR series are different from the VH and VL sequences of the anti-HER2 scFv site in the PE series.
The sequence of the construct TR-4 is disclosed in SEQ ID NO: 64.
The sequence of the construct TR-7 is disclosed in SEQ ID NO: 65.
The sequence of the construct PE-1 is disclosed in SEQ ID NO: 66.
The sequence of the construct PE-3 is disclosed in SEQ ID NO: 67.
The constructs were expressed and run on a non-reducing SDS-PAGE gel.
In this example variants of bispecific antibodies capable of binding CD20 and DOTAM was generated. The SADA domain was the domain disclosed in SEQ ID NO: 5.
Two versions of the Anti-CD20-Anti-DOTAM-SADA conjugate were generated:
The sequence of the construct Ri-12 is disclosed in SEQ ID NO: 68.
The sequence of the construct Ri-13 is disclosed in SEQ ID NO: 69.
The two constructs were expressed and run on a non-reducing SDS-PAGE gel, as shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| PA 2021 70622 | Dec 2021 | DK | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/DK2022/050280 | 12/14/2022 | WO |
