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WO2024240690A2 - Domaines variables d'anticorps et anticorps présentant une immunogénicité réduite - Google Patents

Domaines variables d'anticorps et anticorps présentant une immunogénicité réduite Download PDF

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Publication number
WO2024240690A2
WO2024240690A2 PCT/EP2024/063788 EP2024063788W WO2024240690A2 WO 2024240690 A2 WO2024240690 A2 WO 2024240690A2 EP 2024063788 W EP2024063788 W EP 2024063788W WO 2024240690 A2 WO2024240690 A2 WO 2024240690A2
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seq
nos
mutations
variants
framework regions
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WO2024240690A3 (fr
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Stefan Warmuth
Maria JOHANSSON
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Numab Therapeutics AG
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Numab Therapeutics AG
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • the present invention relates to antibody variable domains, which exhibit a reduced binding to anti-drug antibodies (ADA), to antibodies comprising one or more of said antibody variable domains, and to pharmaceutical compositions comprising said antibodies.
  • the present invention further relates to nucleic acids encoding said antibody variable domains or said antibodies, vector(s) comprising said nucleic acids, host cell(s) comprising said nucleic acids or said vector(s), and a method of producing said antibody variable domains or said multispecific antibodies.
  • the present invention relates to a method for generating said antibody variable domains and antibodies.
  • ADAs anti-drug antibodies
  • ADAs can be antibodies, which are already present in human serum (so called pre-existing ADAs) and/or antibodies, which are formed during the course of the therapy, also referred to as treatment-emerging ADAs.
  • ADA-binding and/or of treatment-emerging ADA formation can be significantly enhanced for therapeutic antibodies that comprise or are built of portions of a naturally occurring human antibody, e. g. Fab or Fv antibody fragments. It is believed that one of the main reasons for this increase in ADA-binding and ADA-formation is that in antibody fragments, typically a significant number of amino acids that are formerly shielded by the 119523P877PC 17.05.2024 Numab Therapeutics AG contact to other antibody portions or domains, become exposed to the solvent and are present to the immune system as potential epitopes.
  • ADA-binding and/or of treatment-emerging ADA formation can also be enhanced for therapeutic antibodies that comprise complementarity- determining regions (CDRs) that have been obtained from non-human species, such as from mouse or rabbit, because CDRs derived from non-human antibody repertoires represent potential B-cell and/or T-cell epitopes.
  • CDRs complementarity- determining regions
  • This risk for ADA-binding and ADA-formation applies to all types of antibodies, to antibody fragments as well as to full-length antibodies or IgG-based antibodies.
  • antibody responses in patients are dependent on the presence of both B-cell epitopes and T-cell epitopes.
  • a B-cell receptor When a B-cell receptor recognizes and binds an antigen such as an administered therapeutic antibody, the antigen is internalized into the B cell by receptor-mediated endocytosis and undergoes proteolytic processing. The resulting peptides are subsequently presented by MHC class II molecules. Upon recognition of the T cell epitope by a T helper cell, the latter stimulates the corresponding B cells to proliferate and differentiate into antibody producing plasma cells. [0007] Several strategies have been provided in the prior art to further lower the response of the immune system of a patient to the administered antibodies.
  • WO 2002/066514 discloses immunogenically modified fusion proteins, which exhibit reduced immunogenicity relative to their non-modified parent fusion protein, when exposed to the immune system of a given species, by having a reduced number of T-cell epitopes within their amino acid sequence.
  • the T-cell epitopes are specified therein to be peptide sequences able to bind to MCH class II molecule binding groups.
  • WO 2002/066514 (A2) further discloses a computational method that profiles the likelihood of peptide regions to contain T-cell epitopes and suggests modifying said peptide regions to potentially alter the MHC Class II binding characteristics of the protein containing them.
  • WO 2002/079232 discloses a method for reducing the immunogenicity of a fusion protein by first identifying a candidate T-cell epitope within a junction region spanning a fusion junction of a fusion protein; and then changing an amino acid within the junction region to reduce the ability of the candidate T-cell epitope to interact with a T cell receptor.
  • T-cell hot spots several computational methods for the identification of amino acid stretches within an antibody that may potentially bind to MHC molecules
  • antibody variable domains provide a high stability, when integrated in the final antibody format, which would allow their application in the construction of stable antibody fragments and fragment-based multispecific antibodies suitable for therapeutic development. It would further be desirable to have a generally applicable method at hand that allows the reliable design and production of said antibody variable domains.
  • SUMMARY OF THE INVENTION It is an object of the present invention to provide antibody variable domain variants, which generally exhibit reduced immunogenicity, more specifically, antibody variable domain variants that are significantly less recognized by MHC class II molecules, i. e. have no predicted T-cell epitopes or at least a significantly reduced number of predicted T-cell epitopes, when compared to their unmodified variants.
  • antibody variable domains which comprise a light chain complementarity-determining region 2 (LCDR2) that has a glycine (G) at amino acid position 67 and a glycine (G) at amino acid position 68 (AHo numbering), hereinafter also referred to as “GG variants” or “GG motif”, exhibit a significantly lower predicted binding to MHC class II proteins, when compared to their original versions that do not comprise said GG motif.
  • GG variants a light chain complementarity-determining region 2
  • AHo numbering glycine-determining region 2
  • the present invention relates to an antibody variable domain, which binds to a target antigen, comprising: (i) a variable heavy chain (VH) comprising from N-terminus to C-terminus, the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW designates a heavy chain framework region, and each HCDR designates a heavy chain complementarity-determining region; and (ii) a variable light chain (VL), wherein the variable light chain comprises, from N-terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, wherein each LFW designates a light chain framework region, and each LCDR designates a light chain complementarity-determining region; wherein said LCDR2 is defined by light chain amino acid positions 58 to 72 according to the AHo numbering scheme, resulting in a maximum length of 15 amino acids, and wherein
  • the present invention relates to an antibody comprising one or more antibody variable domains of the present invention.
  • the present invention relates to a nucleic acid or two nucleic acids encoding the antibody variable domain or the antibody of the present invention.
  • the present invention relates to a vector or two vectors comprising the nucleic acid or the two nucleic acids of the present invention.
  • the present invention relates to a host cell or host cells comprising the vector or the two vectors of the present invention.
  • the present invention relates to a method for producing the antibody variable domain of the present invention or the antibody of the present invention, comprising (i) providing the nucleic acid or the two nucleic acids of the present invention, or the vector or the two vectors of the present invention, expressing said nucleic acid or said two nucleic acids, or said vector or vectors, and collecting said antibody variable domain or said antibody from the expression system, or (ii) providing a host cell or host cells of the present invention, culturing said host cell or said host cells; and collecting said antibody variable domain or said antibody from the cell culture.
  • the present invention relates to a pharmaceutical composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier.
  • the present invention relates to the pharmaceutical composition of the present invention for use as a medicament.
  • the present invention relates to a method for generating a modified antibody variable domain, wherein the method comprises the steps of 1) providing an unmodified antibody variable domain, which binds to a target antigen, comprising: (i) a variable heavy chain (VH) comprising from N-terminus to C-terminus, the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW designates a heavy chain framework region, and each HCDR designates a heavy chain complementarity-determining region; and (ii) a variable light chain (VL), wherein the variable light chain comprises, from N- terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3- LFW4, wherein each LFW designates a light chain framework region, and each LCDR designates
  • the present invention relates to a method for generating a modified antibody from an unmodified antibody, the method comprises the step of 1) selecting one or more antibody variable domains comprised in said unmodified antibody, which do not comprise a glycine (G) at both amino acid position 67 and 68 (AHo numbering) in the LCDR2, for modification; 2) introducing into each of said one or more antibody variable domains selected in step 1) a glycine (G) at amino acid position 67 and/or a glycine (G) at amino acid position 68 (AHo numbering) in the LCDR2, such that there is a glycine (G) at both amino acid positions 67 and 68 (AHo numbering) in the LCDR2.
  • An antibody variable domain which binds to a target antigen, comprising: a. a variable heavy chain (VH) comprising from N-terminus to C-terminus, the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW designates a heavy chain framework region, and each HCDR designates a heavy chain complementarity-determining region; and b.
  • VH variable heavy chain
  • variable light chain wherein the variable light chain comprises, from N- terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3- LFW4, wherein each LFW designates a light chain framework region, and each LCDR designates a light chain complementarity-determining region;
  • said LCDR2 is defined by light chain amino acid positions 58 to 72 according to the AHo numbering scheme, resulting in a maximum length of 15 amino acids, and wherein said LCDR2 has a glycine (G) at amino acid position 67 and a glycine (G) at amino acid position 68 (AHo numbering), particularly wherein said light chain complementarity-determining region LCDR2 consists of seven amino acids; and wherein said light chain complementarity-determining region LCDR2, i.
  • amino acid positions 58, 67, 68, 69, 70, 71, 72 does not have a sequence selected from LGGNRAA (SEQ ID NO: 665) and RGGERVS (SEQ ID NO: 666). 2.
  • variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 and/or said variable light chain framework regions LFW1, LFW2, LFW3 and LFW4 are selected from a) human antibody framework regions; and 119523P877PC 17.05.2024 Numab Therapeutics AG b) human antibody framework regions comprising a total of 1 to 20, particularly 1 to 15, particularly 1 to 10, amino acid positions where the amino acids are taken from a non- human antibody, in particular from a lagomorph or rodent antibody, in particular from a rabbit or mouse antibody, in particular from a rabbit antibody (back mutations), said non-human antibody representing the source of the CDR sequences comprised in the antibody variable domain. 3.
  • variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 and/or said variable light chain framework regions LFW1, LFW2, LFW3 and LFW4 are selected from framework regions of an antibody that has been tested in clinical trials.
  • said variable variable domain of any one of the items 1 to 3 wherein a. said variable light chain framework regions LFW1, LFW2, LFW3 and LFW4 together are selected from a human antibody V ⁇ framework or a human antibody V ⁇ framework, in particular a human antibody V ⁇ framework; or b. said variable light chain framework regions LFW1, LFW2 and LFW3 are selected from a human antibody V ⁇ framework, and said variable light chain framework region LFW4 is selected from a human antibody V ⁇ framework. 5.
  • the antibody variable domain of item 4, wherein said human antibody V ⁇ framework is selected from the V ⁇ framework subtypes V ⁇ 1, V ⁇ 2, V ⁇ 3 and V ⁇ 4. 6. The antibody variable domain of item 4, wherein said human antibody V ⁇ framework is of the V ⁇ framework subtype V ⁇ 1. 7. The antibody variable domain of item 4, wherein said human antibody V ⁇ framework is selected from the V ⁇ framework subtypes V ⁇ 1, V ⁇ 2 and V ⁇ 3. 8.
  • variable light chain framework region LFW4 when selected from a human antibody V ⁇ framework, has a sequence selected from a) any one of SEQ ID NOs: 527, 528, 529, 530, 531, 532, 533, 534 and 535; or b) any one of SEQ ID NOs: 527, 528, 529, 530, 531, 532, 533, 534 and 535, which has one or two mutations, particularly one mutation, particularly one or two mutations at one or two positions selected from positions 141, 144, 145, 146 and 147 (AHo numbering), more particularly one mutation at position 141 (AHo numbering).
  • variable light chain framework regions LFW1, LFW2, LFW3 and LFW4 are selected from a. the combination of framework regions LFW1, LFW2, LFW3 and LFW4 (i. e. the residues corresponding to the non-italicized residues of SEQ ID NO: 3 or of the VL sequences shown in Table 2) of any one of the SEQ ID NOs: 304, 309, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525 and 526; and b. the combination of framework regions LFW1, LFW2, LFW3 and LFW4 (i. e.
  • variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from the human VH framework subtypes VH1a, VH1b, VH3, VH4, VH5 and VH6. 12. The antibody variable domain of any one of the preceding items, wherein said variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from the human VH framework subtypes VH1a, VH1b, VH3 and VH4. 13. The antibody variable domain of any one of the preceding items, wherein said variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are of the human VH framework subtype VH3. 14.
  • variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from a. the combination of framework regions HFW1, HFW2, HFW3 and HFW4 (i. e. the residues corresponding to the non-italicized residues of SEQ ID NO: 1 or of the VH sequences shown in Table 2) of any one of the SEQ ID NOs: 302, 306, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513 and 514; and b. the combination of framework regions HFW1, HFW2, HFW3 and HFW4 (i. e.
  • variable light chain framework regions LFW1, LFW2 and LFW3 are selected from a human antibody V ⁇ framework, in particular are of the V ⁇ framework subtype V ⁇ 1;
  • said variable light chain framework region LFW4 is selected from a human antibody V ⁇ framework, in particular has a sequence selected from a. any one of SEQ ID NOs: 527, 528, 529, 530, 531, 532, 533, 534 and 535; or b.
  • variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are of the human VH framework subtype VH3. 16. The antibody variable domain of item 1, wherein (i) said variable light chain framework regions LFW1, LFW2, LFW3 and LFW4 are selected from a.
  • framework regions LFW1, LFW2, LFW3 and LFW4 i. e. the residues corresponding to the non-italicized residues of SEQ ID NO: 3 or of the VL sequences shown in Table 2 of any one of the SEQ ID NOs: 304, 309, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525 and 526; and b. the combination of framework regions LFW1, LFW2, LFW3 and LFW4 (i. e.
  • variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 are selected from a. the combination of framework regions HFW1, HFW2, HFW3 and HFW4 (i. e.
  • LCDR1 and LCDR3 are selected from: (i) LCDR1 and LCDR3 regions of said mammalian antibody; or (ii) LCDR1 and LCDR3 regions of said antibody that has been tested in clinical trials; or (iii) LCDR1 and LCDR3 regions of an antibody according to (i) or (ii), wherein at least in one of said LCDR1 and LCDR3 regions, independently from each other, one, two or three amino acid positions, in particular one or two amino acid positions, have been altered. 19.
  • said light chain complementarity-determining region LCDR2 has one or more of the following features: (a) has a lysine (K), leucine (L), methionine (M) or arginine (R), in particular a lysine (K), methionine (M) or leucine (L), in particular a leucine (L), at amino acid position 70 (AHo numbering); (b) has a sequence selected from X1GGX2X3X4X5 (SEQ ID NO: 536), wherein X 1 is selected from lysine (K), arginine (R), serine (S), threonine (T), alanine (A), leucine (L), glycine (G), glutamine (Q), glutamate (E), tyrosine (Y) and aspartate (D); in particular from lysine (K), arginine (R), serine (S), tyrosine (K), arginine (R), serine (S),
  • variable light chain of said antibody variable domain comprises no 15mer peptide stretch having a median percentile rank, as determined from the predicted individual binding strengths (scoring ranks) of said 15mer peptide to each one of the 27 MHC class II alleles that are most common in the general population using the algorithm of NetMHCIIpan-3.1, of less than 13, particularly less than 14, particularly less than 15, particularly less than 16, particularly less than 17, particularly less than 18, particularly less than 19, particularly less than 20. 24.
  • variable variable domain of any one of the preceding items wherein the variable heavy chain of said antibody variable domain comprises no 15mer peptide stretch having a median percentile rank, as determined from the predicted individual binding strengths (scoring ranks) of said 15mer peptide to each one of the 27 MHC class II alleles that are most common in the general population using the algorithm NetMHCIIpan-3.1, of less than 13, particularly less than 14, particularly less than 15, particularly less than 16, particularly less than 17, particularly less than 18, particularly less than 19, particularly less than 20. 119523P877PC 17.05.2024 Numab Therapeutics AG 25.
  • the antibody variable domain of any one of the preceding items, wherein said antibody variable domain, when being in scFv format is further characterized by one or more of the following features: a.
  • Tm melting temperature
  • DSF differential scanning fluorimetry
  • KD dissociation constant
  • variable domain as defined in any one of items 1 to 26, wherein the variable heavy chain (VH) comprises a serine (S) at amino acid position 101 and a lysine (K) at amino acid position 146 (AHo numbering).
  • VH variable heavy chain
  • K lysine
  • An antibody comprising one or more antibody variable domains as defined in any one of items 1 to 27. 29.
  • the IgG region is selected from the IgG subclasses IgG1 and IgG4. 33. The antibody of any one of items 28 and 29, wherein said antibody does not comprise an immunoglobulin Fc region. 34.
  • the antibody of item 33 wherein said antibody is in a format selected from the group consisting of: a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a circular dimeric scDb (CD-scDb), a tandem tri-scFv, a tribody (Fab-(scFv) 2 ), a Fab-Fv 2 , a triabody, an scDb-scFv, a tetrabody, a didiabody, a tandem-di-scFv and a MATCH.
  • Fab-(scFv) 2 Fab-(scFv) 2
  • Fab-Fv 2 Fab-Fv 2
  • the antibody of item 35 wherein said antibody is in a scDb-scFv, a triabody, a tetrabody or a MATCH format, in particular wherein said antibody is in a MATCH or scDb-scFv format, more particularly wherein said antibody is in a MATCH format, more particularly a MATCH3 or a MATCH4 format. 37.
  • a nucleic acid or two nucleic acids encoding the antibody variable domain of any one of items 1 to 26, or the antibody of any one of items 28 to 37.
  • a vector or two vectors comprising the nucleic acid or the two nucleic acids of item 38.
  • a host cell or host cells comprising the vector or the two vectors of item 39. 41.
  • a method for producing the antibody variable domain of any one of items 1 to 27, or the antibody of any one of items 28 to 37 comprising (i) providing the nucleic acid or the two nucleic acids of item 38, or the vector or the two vectors of item 39, expressing said nucleic acid sequence or nucleic acids, or said vector or vectors, and collecting said antibody variable domain or said antibody from the expression system, or (ii) providing a host cell or host cells of item 40, culturing said host cell or said host cells; and collecting said antibody variable domain or said antibody from the cell culture.
  • a pharmaceutical composition comprising the antibody of any one of items 28 to 37 and a pharmaceutically acceptable carrier. 43.
  • a method for generating a modified antibody variable domain from an unmodified antibody variable domain wherein said unmodified antibody variable domain binds to a target antigen and comprises: (i) a variable heavy chain (VH) comprising from N-terminus to C-terminus, the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW designates a heavy chain framework region, and each HCDR designates a heavy chain complementarity-determining region, and (ii) a variable light chain (VL), wherein the variable light chain comprises, from N- terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3- LFW4, wherein each LFW designates a light chain framework region, and each LCDR designates a light chain complementarity-determining region, wherein the light chain complementarity-determining region
  • a method for generating a modified antibody variable domain comprising the steps of 1) providing an unmodified antibody variable domain, which binds to a target antigen, comprising: (i) a variable heavy chain (VH) comprising from N-terminus to C-terminus, the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW designates a heavy chain framework region, and each HCDR designates a heavy chain complementarity-determining region; and (ii) a variable light chain (VL), wherein the variable light chain comprises, from N- terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3- LCDR3-LFW4, wherein each LFW designates a light chain framework region, and each LCDR designates a light chain complementarity-determining region, wherein the light chain complementarity-determining region LCDR2 does not comprise a glycine (G) at both amino acid position 67 and
  • modified antibody variable domain comprises a decreased number of 15mer peptide stretches that exhibit a median percentile rank of less than 20, as determined from the predicted individual binding strengths (scoring ranks) of said 15mer peptide to each one of the 27 MHC class II alleles that are most common in the general population using the algorithm NetMHCIIpan-3.1, when compared to the respective unmodified antibody variable domain.
  • the LCDR2 of the unmodified antibody variable domain comprises amino acid residues at positions 58, 67, 68, 69, 70, 71 and 72 (AHo numbering).
  • the light chain complementarity- determining region LCDR2 of the unmodified antibody variable domain has a sequence selected from X 1 GGX 2 X 3 X 4 X 5 (SEQ ID NO: 536), wherein X 1 , X 2 , X 3 , X 4 and X 5 correspond 119523P877PC 17.05.2024 Numab Therapeutics AG to amino acid positions 58, 69, 70, 71 and 72 (AHo numbering), and wherein X1, X2, X3, X 4 and X 5 of LCDR2 are selected from (i) the corresponding positions of an LCDR2 region of a mammalian antibody, in particular of an LCDR2 region of a lagomorph, rodent or human antibody, in particular of an LCDR2 region of a rabbit, mouse or human antibody, in particular of an LCDR2 region of a rabbit antibody; or (ii) the corresponding positions of an LCDR2 region of an antibody that has been tested in clinical trials
  • the LCDR2 of the unmodified antibody variable domain comprises: (i) an alanine (A), threonine (T), serine (S) or glycine (G) at position 67 (AHo numbering); (ii) a serine (S), tyrosine (Y) or phenylalanine (F) at position 68 (AHo numbering); and optionally (iii) a lysine (K), threonine (T), serine (S), isoleucine (I), aspartate (D), asparagine (N), phenylalanine (F) or tyrosine (Y) at position 69 (AHo numbering); (iv) an alanine (A), glycine (G), valine (V), threonine (T), proline (P), tyrosine (Y), aspartate (D) or glutamate (E) at position 71 (AHo numbering
  • the LCDR2 of the unmodified antibody variable domain comprises: (i) an alanine (A) or threonine (T) at position 67 (AHo numbering); (ii) a serine (S) or tyrosine (Y) at position 68 (AHo numbering); and optionally (iii) a lysine (K), threonine (T), serine (S), aspartate (D), asparagine (N) or tyrosine (Y) at position 69 (AHo numbering); (iv) an alanine (A), glycine (G), threonine (T) or glutamate (E) at position 71 (AHo numbering).
  • the LCDR2 of the unmodified antibody variable domain comprises: (i) lysine (K), arginine (R), serine (S), threonine (T), alanine (A), leucine (L), glycine (G), glutamine (Q), glutamate (E), tyrosine (Y) or aspartate (D) at position 58 (AHo numbering); (ii) an alanine (A), threonine (T), serine (S) or glycine (G) at position 67 (AHo numbering); (iii) a serine (S), tyrosine (Y) or phenylalanine (F) at position 68 (AHo numbering); 119523P877PC 17.05.2024 Numab Therapeutics AG (iv) a lysine (K), threonine (T), serine (S), isoleucine (I), aspartate (D),
  • the LCDR2 of the unmodified antibody variable domain comprises: (i) a lysine (K), arginine (R), serine (S), tyrosine (Y), glycine (G) or aspartate (D) at position 58 (AHo numbering); (ii) an alanine (A) or threonine (T) at position 67 (AHo numbering); (iii) a serine (S) or tyrosine (Y) at position 68 (AHo numbering); (iv) a lysine (K), threonine (T), serine (S), aspartate (D), asparagine (N) or tyrosine (Y); (v) a leucine (L) at position 70 (AHo numbering); (vi) an alanine (A), glycine (G), threonine (T) or glutamate (E) at position 71 (AHo
  • any one of the items 44 to 46, wherein the light chain complementarity- determining region LCDR2 of the modified antibody variable domain has a sequence selected from X1GGX2X3X4X5 (SEQ ID NO: 536), wherein X1, X2, X3, X4 and X5 correspond to amino acid positions 58, 69, 70, 71 and 72 (AHo numbering), and wherein said X1, X2, X 3 , X 4 and X 5 of LCDR2 are selected from: (i) the corresponding positions of an LCDR2 region of a mammalian antibody, in particular of an LCDR2 region of a lagomorph, rodent or human antibody, in particular of an LCDR2 region of a rabbit, mouse or human antibody, in particular of an LCDR2 region of a rabbit antibody; or (ii) the corresponding positions of an LCDR2 region of an antibody that has been tested in clinical trials; or (iii) the corresponding positions of an LCDR2 region of an antibody according to
  • any one of items 46 to 54 wherein in said modified antibody variable domain, the number of 15mer peptide stretches that exhibit a median percentile rank of less than 20, as determined from the predicted individual binding strengths (scoring ranks) of said 15mer peptide to each one of the 27 MHC class II alleles that are most common in the general population using the algorithm NetMHCIIpan-3.1, is decreased by at least 50%, particularly by at least 60%, particularly by at least 70%, particularly by at least 80%, particularly by at least 90%, when compared to the respective unmodified antibody variable domain. 58.
  • any one of items 44 to 57 wherein the method comprises that additional step of 3) introducing into the variable heavy chain (VH) of the unmodified antibody variable domain provided in step 1) a serine (S) at amino acid position 101 and a lysine (K) at amino acid position 146 (AHo numbering). 59.
  • a method for generating a modified antibody from an unmodified antibody comprises the steps of 1) selecting one or more antibody variable domains comprised in said unmodified antibody, which do not comprise a glycine (G) at both amino acid position 67 and 68 (AHo numbering) in the LCDR2, for modification; 2) introducing into each of said one or more antibody variable domains selected in step 1) a glycine (G) at amino acid position 67 and/or a glycine (G) at amino acid position 68 (AHo numbering) in the LCDR2, such that there is a glycine (G) at both amino acid positions 67 and 68 (AHo numbering) in the LCDR2, wherein said LCDR2 is defined by light chain amino acid positions 58 to 72 according to the AHo numbering scheme, resulting in a maximum length of 15 amino acids.
  • the modified antibody comprises a decreased number of 15mer peptide stretches that exhibit a median percentile rank of less than 20, as determined from the predicted individual binding strengths (scoring ranks) of said 15mer peptide to each one of the 27 MHC class II alleles that are most common in the general population using the algorithm NetMHCIIpan-3.1, when compared to the respective unmodified antibody. 61.
  • FIG.1 shows a median percentile rank plot of the VL region of the anti-PDL1 scFv PRO2230, calculated with NetMHCIIpan-3.1.
  • FIG.2 shows median percentile rank plots of the VL regions of the multispecific anti-PDL1xCD137xhSA antibody PRO1480 (A) and of a GG variant of PRO1480 (B), calculated with NetMHCIIpan-3.1. The plots for VL regions of the PDL1, CD137 (4-1BB) and hSA binding domains are superimposed.
  • the dashed line indicates the median percentile rank 20. A median percentile rank of less than 20 is deemed to indicate significant binding to MHC class II proteins.
  • FIG.3 shows median percentile rank plots of the VL regions of 206 therapeutic antibodies (A) and of their corresponding GG variants (B), calculated with NetMHCIIpan-3.1. All VL region plots are superimposed. The dashed line indicates the median percentile rank 20. A median percentile rank of less than 20 is deemed to indicate significant binding to MHC class II proteins.
  • FIG.4 shows the absorption levels of pre-existing and treatment-emerging ADAs against PRO1480 in five patients at various time points, determined with the ELISA-based pre-existing ADA-binding assay described in Example 3. Absorption levels were measured at 450 nm and are given in A.U.
  • FIG.5 shows the inhibition of the absorption signal after spiking each serum with the respective molecule (MATCH-3 molecules PRO1480, PRO4180, PRO4181: 150 nM spiking; scFv PRO2230: 450 nM spiking), determined with the ELISA-based ADA-binding assay described in Example 3.
  • the reduction in the signal for the spiked sera vs. non-spiked sera has to be greater than 30% (>30% inhibition) in order to be evaluated as specific.
  • antibody variable domains which comprise a light chain complementarity-determining region 2 (LCDR2) that has a glycine (G) at amino acid position 67 and a glycine (G) at amino acid position 68 (AHo numbering), hereinafter also referred to as “GG variants” or “GG motif”, exhibit a significantly lower predicted binding to (human) MHC class II proteins, when compared to their original versions that do not comprise said GG motif.
  • GG variants a light chain complementarity-determining region 2
  • antibody variable domains which exhibit low immunogenicity. More specifically, it would be desirable to have antibody variable domains at hand, which exhibiting low immunogenicity, in particular with regard to reduced binding to MHC class II proteins, and which can generally be applied in the construction of antibody fragments. It is furthermore desirable that these antibody variable domains provide a high stability, when integrated in the final antibody format, which would allow their application in the construction of stable antibody fragments and fragment-based multispecific antibodies suitable for therapeutic development.
  • the antibody variable domain of the present invention could be successfully used for the construction of several highly stable and functional antibody binding domains, such as Fab fragments and scFvs, that can readily be used in the construction of multispecific antibodies.
  • Fab fragments and scFvs highly stable and functional antibody binding domains
  • scFvs highly stable and functional antibody binding domains
  • the universal applicability of this modification was tested with a set of 206 sequences of antibodies that have been clinically tested, i. e. clinically approved and clinical phase 1–3 antibody therapeutics. These sequences have been published in Marks et al.
  • the present invention relates to an antibody variable domain, which binds to a target antigen, comprising: (i) a variable heavy chain (VH) comprising from N-terminus to C-terminus, the regions HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4, wherein each HFW designates a heavy chain framework region, and each HCDR designates a heavy chain complementarity-determining region; and 119523P877PC 17.05.2024 Numab Therapeutics AG (ii) a variable light chain (VL), wherein the variable light chain comprises, from N-terminus to C-terminus, the regions LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4, wherein each LFW designates a light chain framework region, and each LCDR designates a light chain complementarity-determining region; wherein said LCDR2 is defined by light chain amino acid positions 58 to 72 according to the AHo numbering scheme, resulting in
  • antibody and the like, as used herein, includes whole antibodies or single chains thereof; and any antigen-binding variable domain (i. e. “antigen-binding portion”) or single chains thereof; and molecules comprising antibody CDRs, VH regions or VL regions (including without limitation multispecific antibodies).
  • a naturally occurring “whole antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), flanked by regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e. g., effector cells) and the first component (Clq) of the classical complement system.
  • the term “antibody variable domain”, as used herein, refers to one or more parts of an intact antibody that have the ability to specifically bind to a given antigen (e.
  • This can be any antigen- binding fragment (i. e. “antigen-binding portion”) of an intact antibody or single chains thereof; and molecules comprising antibody CDRs, VH regions or VL regions.
  • Fab a single chain Fab
  • scFab single chain Fab
  • dsFv disulfide stabilized Fv fragment
  • scFv single chain Fv fragment having an additional light chain constant domain (CL) fused to it
  • the antibody variable domain of the present invention is selected from a Fab, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) and an scFv fragment.
  • the antibody variable domain of the present invention is a Fab.
  • the antibody variable domain of the present invention is a single- chain Fv fragment (scFv).
  • the VL and VH domains of the scFv fragment are stabilized by an interdomain disulfide bond, in particular said VH domain comprises a single cysteine residue in position 51 (AHo numbering) and said VL domain comprises a single cysteine residue in position 141 (AHo numbering).
  • CDRs Complementarity Determining Regions
  • the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52- 56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).
  • the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50- 56 (LCDR2), and 89-97 (LCDR3) in human VL.
  • the CDR amino acid residues in the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2) and 93-102 (HCDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering according to “Kabat”).
  • the CDRs of an antibody can be determined using the program IMGT/DomainGap Align. [0047]
  • the numbering system suggested by Honegger & Plückthun (“AHo”) is used (Honegger & Plückthun, J. Mol.
  • CDRs are defined as CDRs according to AHo numbering scheme: LCDR1 (also referred to as CDR- L1): L24-L42; LCDR2 (also referred to as CDR-L2): L58-L72; LCDR3 (also referred to as CDR-L3): L107-L138; HCDR1 (also referred to as CDR-H1): H27-H42; HCDR2 (also referred to as CDR-H2): H57-H76; HCDR3 (also referred to as CDR-H3): H108-H138.
  • LCDR1 also referred to as CDR- L1
  • LCDR2 also referred to as CDR-L2
  • LCDR3 also referred to as CDR-L3
  • H108-H138 H108-H138.
  • LFW1-LFW4 light chain frameworks 1 to 4
  • heavy chain frameworks 1 to 4 HFW1- HFW4
  • AHo numbering scheme LFW1: L1-L23; LFW2: L43-L57; LFW3: L73-L106; LFW4: L139-L149; HFW1: H1-H26; HFW2: H43-H56; HFW3: H77-H107; HFW4: H139-H149.
  • the numbering system according to Honegger & Plückthun takes the length diversity into account that is found in naturally occurring antibodies, both in the different VH and VL subfamilies and, in particular, in the CDRs, and provides for gaps in the sequences.
  • a given antibody variable domain usually not all positions 1 to 149 will be occupied by an amino acid residue.
  • said framework (FW) regions and said CDR regions comprised in the antibody fragments of the present invention have maximum length corresponding to the above residue numbering under the Aho numbering system.
  • the maximum sequence length is limited to 15 amino acids (resulting from the Aho numbering L58-L72). Consequently, artificial antibody variable domains that have a longer amino acid stretches in one or more of said FW regions and/or said CDR regions, e.g. having an LCDR2 that is larger than 15 amino acids, are not considered as antibody variable domains according to the present invention.
  • the antibody variable domains of the present invention have an LCDR2, which has a length of at maximum 14, particularly of at maximum 12, particularly of at maximum 10, particularly of at maximum 9, particularly of at maximum 8, particularly of at maximum 7 amino acids.
  • the antibody variable domains of the present invention have an LCDR2 that consists of seven amino acids.
  • binding to refers to the ability of an individual antibody to react with an antigenic determinant. However, this does not exclude that said individual antibody can for example also react with homologues of said antigenic determinant (e. g. with antigen determinants from other species) or with other antigen determinants belonging to the same protein family.
  • binding specificity refers to the ability of an individual antibody to react with one antigenic determinant and not with a different antigenic 119523P877PC 17.05.2024 Numab Therapeutics AG determinant.
  • the term “specifically binds to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • an antibody that specifically binds to a target is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets.
  • these terms do not exclude that said individual antibody can bind with comparable affinity to the same antigen determinants from different species.
  • “specific binding” is referring to the ability of the antibody to discriminate between the target of interest and an unrelated molecule, as determined, for example, in accordance with specificity assay methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA, RIA, ECL, IRMA, SPR (Surface plasmon resonance) tests and peptide scans.
  • a standard ELISA assay can be carried out. The scoring may be carried out by standard color development (e. g. secondary antibody with horseradish peroxide and tetramethyl benzidine with hydrogen peroxide).
  • the reaction in certain wells is scored by the optical density, for example, at 450 nm.
  • an SPR assay can be carried out, wherein at least 10-fold, particularly at least 100-fold difference between a background and signal indicates on specific binding.
  • determination of binding specificity is performed by using not a single reference molecule, but a set of about three to five unrelated molecules, such as milk powder, transferrin or the like.
  • the antibody variable domains of the present invention bind to a target antigen, which can be any target antigen.
  • target antigens include, but are not limited to: a transmembrane molecule; a receptor; a ligand; a growth factor; a growth hormone; a clotting factor; an anti-clotting factor; a plasminogen activator; a serum albumin; a receptor for a hormone or a growth factor; a neurotrophic factor; a nerve growth factor; a fibroblast growth factor; a CD protein; an interferon; a colony stimulating factor (CSF); an interleukin (IL); a T-cell receptor; a T-cell co-stimulatory receptor, such as CD137; a surface membrane protein; a viral protein; a tumor associated antigen; an integrin or an interleukin; VEGF; a renin; a human growth hormone; a bovine growth hormone; a growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; a lipoprotein; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; pro
  • TAA tumor-associated antigen
  • a TAA is an antigen that is preferentially expressed on a tumor cell when compared to non-tumor cells, particularly wherein expression of the TAA on a tumor cell is at least more than 5-fold, at least more than 10-fold, at least more than 20-fold, at least more than 50- fold, or at least more than 100-fold higher than on non-tumor cells from the same organism or patient.
  • tumor associated antigen targets include, but are not limited to: ADRB3, AFP, ALK, BCMA, beta human chorionic gonadotropin, CA-125 (MUC16), CAIX, CD123, CD133, CD135, CD135 (FLT3), CD138, CD171, CD19, CD20, CD22, CD24, CD276, CD33, CD33, CD38, CD44v6, CD79b, CD97, CDH3 (cadherin 3), CEA, CEACAM6, CLDN6, CLEC12A (CLL1), CSPG4, CYP1B1, EGFR, EGFRvlll, EpCAM, EPHA2, Ephrin B2, ERBBs (e. g.
  • ERBB2 Her2/neu (HER2), HLA-A2, HMWMAA, HPV E6 or E7, human telomerase reverse transcriptase, IL-11Ra, IL-13Ra2, intestinal carboxyl esterase, KIT, Legumain, LewisY, LMP2, Ly6k, MAD-CT-1, MAD-CT-2, ML-IAP, MN-CA IX, MSLN, MUC1, mut hsp 70-2, NA- 17, NCAM, neutrophil elastase, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, PANX3, PDGFR-beta, PLAC1, Polysia
  • Preferred examples are: CD138, CD79b, TPBG (5T4), HER2, MSLN, MUC1, CA- 125 (MUC16), PSMA, BCMA, CD19, EpCAM, CLEC12A (CLL1), CD20, CD22, CEA, CD33, EGFR, GPC3, CD123, CD38, CD33, CD276, CDH3 (cadherin 3), FGFR1, SSTR2, CD133, EPHA2, HLA-A2, IL13RA2, ROR1, CEACAM6, CD135, GD-2, GA733, CD135 (FLT3), CSPG4 and TAG-72.
  • the framework regions HFW1, HFW2, HFW3 and HFW4 comprised in the antibody variable domain of the invention are selected from a human VH framework.
  • the framework regions HFW1, HFW2, HFW3 and HFW4 comprised in the antibody variable domain of the invention are selected from the human VH framework subtypes VH1a, VH1b, VH3, VH4, VH5 or VH6, particularly from the human VH framework subtypes VH1a, VH1b, VH3 or VH4.
  • the framework regions HFW1, HFW2, HFW3 and HFW4 are selected from the human VH framework subtype VH3.
  • HFW4 may be selected from a human germline sequence or from the HFW4 sequence of a rearranged human antibody sequence.
  • the terms “belonging to a human VHx framework subtype (or a human antibody V ⁇ /V ⁇ framework)”, “selected from a human VHx framework subtype (or a human antibody V ⁇ /V ⁇ framework)” or “are of the human VHx framework subtype” mean that the framework sequences HFW1 to HFW3 (or LF1 to LFW3) show the highest degree of homology to the consensus sequence of said human antibody VH or VL framework subtype, as determined in Knappik et al., J. Mol. Biol.296 (2000) 57-86 or in WO 2019/057787.
  • the sequences of human VH domains are grouped into seven distinct framework subtypes, i. e. the framework subtypes VH1a, VH1b, VH2, VH3, VH4, VH5 and VH6, herein also referred to as human subfamilies VH1a, VH1b, VH2, VH3, VH4, VH5 and VH6, based on sequence homology to the sequences as shown in Figure 3 of Knappik et al., J. Mol. Biol.296 (2000) 57-86 or in WO 2019/057787.
  • Specific example of VH domains belonging to the VH3 framework subtype are represented by SEQ ID NOs: 496 to 509.
  • VH domain belonging to the VH1a, VH1b, VH4, VH5 and VH6 framework subtypes are represented by SEQ ID NOs: 119523P877PC 17.05.2024 Numab Therapeutics AG 510 - 514.
  • Alternative examples of VH1a, VH1b, VH3 and VH4 sequences, and examples of other VHx sequences, may be found in Knappik et al., J. Mol. Biol.296 (2000) 57-86 or in WO 2019/057787.
  • variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 of the antibody variable domain of the present invention are selected from (i) human antibody framework regions; and (ii) human antibody framework regions comprising a total of 1 to 20, particularly 1 to 15, particularly 1 to 10, amino acid positions where the amino acids are taken from a non- human antibody, in particular from a lagomorph or rodent antibody, in particular from a rabbit or mouse antibody, in particular from a rabbit antibody (back mutations), said non- human antibody representing the source of the CDR sequences comprised in the antibody variable domain.
  • variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 of the antibody variable domain of the present invention are selected from framework regions of antibodies that have been tested in clinical trials.
  • expression “antibodies that have been tested in clinical trials” refers to antibodies that have already been tested in clinical trials as well as to antibodies that are currently in clinical trials. This applies to antibodies that have successfully passed clinical testing as well as to antibodies that failed in clinical testing.
  • the variable heavy chain framework regions HFW1, HFW2, HFW3 and HFW4 of the antibody variable domain of the present invention are selected from the combination of framework regions (i. e.
  • Numab Therapeutics AG non-italicized residues of SEQ ID NO: 1 or of the VH sequences shown in Table 2, i. e. all residues that are not marked as CDR residues) of any one of the SEQ ID NOs: 302, 306, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513 and 514; and from the combination of framework regions (i. e.
  • mutation means, as various non- limiting examples, an addition, substitution or deletion.
  • the VH regions further include VH domains comprising at least positions 5 to 140 (AHo numbering), particularly at least positions 3 to 145, more particularly at least positions 2 to 147 of one of the sequences shown in the SEQ ID NOs: 302, 306, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513 and 514, provided that such VH domains exhibit the functional features defined above in item 25.
  • the variable light chain frameworks LFW1, LFW2, LFW3 and LFW4 of the antibody variable domain of the present invention are selected from a human antibody V ⁇ framework (e. g.
  • a V ⁇ 1, V ⁇ 2, V ⁇ 3 or V ⁇ 4 framework subtype or a human antibody V ⁇ framework (e. g. a V ⁇ 1, V ⁇ 2 or V ⁇ 3 framework subtype), in particular a human antibody V ⁇ framework.
  • the sequences of human VL domains are grouped into four distinct human V ⁇ framework subtypes, i. e. the framework subtypes V ⁇ 1, V ⁇ 2, V ⁇ 3 and V ⁇ 4, and three distinct human V ⁇ framework subtypes, i. e.
  • the variable light chain frameworks LFW1, LFW2, LFW3 and LFW4 of the antibody variable domain of the present invention are of the V ⁇ 1 framework subtype. Specific examples of V ⁇ 1 framework subtypes are represented by SEQ ID NOs: 515, 516, 517 and 518.
  • variable light chain frameworks LFW1, LFW2 and LFW3 are selected from a human antibody V ⁇ framework, preferably a V ⁇ 1 framework subtype, and the variable light chain framework LFW4 is selected from a V ⁇ framework.
  • variable light chain framework LFW4 of the antibody variable 119523P877PC 17.05.2024 Numab Therapeutics AG domain of the present invention is selected from the group consisting of the V ⁇ framework 4 sequences of SEQ ID NOs: 527, 528, 529, 530, 531, 532, 533, 534 and 535.
  • V ⁇ framework 4 sequence of SEQ ID NO: 534 comprises a single cysteine residue at the variable light (VL) chain position 144 (AHo numbering) and is in particular applied in cases where a second single cysteine is present in the corresponding variable heavy (VH) chain, particularly in position 51 (AHo numbering) of VH, for the formation of an inter-domain disulfide bond.
  • the LFW1, LFW2 and LFW3, and optionally also LFW4 if LFW4 is selected from a human antibody V ⁇ framework subtype, are selected from the combination of framework regions LFW1, LFW2, LFW3 and optionally LFW4 (i. e. the residues corresponding to the non-italicized residues of SEQ ID NO: 3 or of the VL sequences shown in Table 2) of any one of the SEQ ID NOs: 515 - 526; and the combination of framework regions LFW1, LFW2, LFW3 and optionally LFW4 (i. e.
  • variable light chain framework regions LFW1, LFW2, LFW3 and LFW4 of the antibody variable domain of the present invention independently from each other, are selected from (i) human antibody framework regions; and (ii) human antibody framework regions comprising a total of 1 to 20, particularly 1 to 15, particularly 1 to 10, amino acid positions where the amino acids are taken from a non- human antibody, in particular from a lagomorph or rodent antibody, in particular from a rabbit or mouse antibody, in particular from a rabbit antibody (back mutations), said non- human antibody representing the source of the CDR sequences comprised in the antibody variable domain.
  • variable light chain framework regions LFW1, LFW2, LFW3 and LFW4 of the antibody variable domain of the present invention are selected from framework regions of antibodies that have been tested in clinical trials.
  • variable light chain frameworks LFW1, LFW2, LFW3 and LFW4 of the antibody variable domain of the present invention are selected from the combination of framework regions LFW1, LFW2, LFW3 and LFW4 (i. e.
  • numab Therapeutics AG residues of SEQ ID NO: 3 or of the VL sequences shown in Table 2) of any one of the SEQ ID NOs: 304, 309, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525 and 526, wherein no more than 5 amino acids, particularly no more than 4 amino acids, particularly no more than 3 amino acids, particularly no more than 2 amino acids, particularly no more than 1 amino acid within the framework regions have been mutated.
  • mutation means, as various non-limiting examples, an addition, substitution or deletion.
  • the VL regions further include VL domains comprising at least positions 5 to 140 (AHo numbering), particularly at least positions 3 to 145, more particularly at least positions 2 to 147 of one of the sequences shown in the SEQ ID NOs: 304, 309, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525 and 526, provided that such VL domains exhibit the functional features defined above in item 25.
  • AHo numbering AHo numbering
  • VL domains comprising at least positions 5 to 140 (AHo numbering), particularly at least positions 3 to 145, more particularly at least positions 2 to 147 of one of the sequences shown in the SEQ ID NOs: 304, 309, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525 and 526, provided that such VL domains exhibit the functional features defined above in item 25.
  • the antibody variable domain of the present invention is in a format selected from a Fab, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a disulfide stabilized Fv fragment (dsFv); and a single chain Fv fragment (scFv).
  • the antibody variable domain of the present invention is selected from a Fab, an Fv fragment and a single-chain Fv fragment (scFv).
  • the VL and VH domains of the scFv fragment are stabilized by an interdomain disulfide bond, in particular said VH domain comprises a single cysteine residue in position 51 (AHo numbering) and said VL domain comprises a single cysteine residue in position 141 (AHo numbering).
  • the light chain complementarity-determining region LCDR2 has a sequence selected from X1GGX2X3X4X5 (SEQ ID NO: 536), wherein X1, X2, X3, X4 and X5 correspond to amino acid positions 58, 69, 70, 71 and 72 (AHo numbering), and wherein said X 1 , X 2 , X 3 , X 4 and X 5 of LCDR2 are selected from: (i) the corresponding positions of an LCDR2 region of a mammalian antibody, in particular of an LCDR2 region of a lagomorph, rodent or human antibody, in particular of an LCDR2 region of a rabbit, mouse or human antibody, in particular of an LCDR2 region of a rabbit antibody; or (ii) the corresponding positions of an LCDR2 region of an antibody that has been tested in clinical trials; or (iii) the corresponding positions of an LCDR2 region of an antibody according to (i) or (ii), wherein
  • the other light chain complementarity- determining regions LCDR1 and LCDR3 and, optionally, also the heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3 are selected from the antibodies defined above for the LCDR2. 119523P877PC 17.05.2024 Numab Therapeutics AG
  • the LCDR2 has a glycine (G) at amino acid position 67, a glycine (G) at amino acid position 68, and a leucine (L) or an arginine (R) at amino acid position 70 (AHo numbering).
  • the LCDR2 has a sequence selected from X1GGX2X3X4X5 (SEQ ID NO: 536), wherein X1 is selected from lysine (K), arginine (R), serine (S), threonine (T), alanine (A), leucine (L), glycine (G), glutamine (Q), glutamate (E), tyrosine (Y) and aspartate (D); in particular from lysine (K), arginine (R), serine (S), tyrosine (Y), glycine (G), and aspartate (D); X2 is selected from lysine (K), threonine (T), serine (S), isoleucine (I), aspartate (D), asparagine (N), phenylalanine (F) and tyrosine (Y); in particular from lysine (K), threonine (T), serine (S), as
  • Non limiting examples of LCDR2 sequences are RGGILAS (SEQ ID NO: 667), KGGTLAS (SEQ ID NO: 668), RGGTLAS (SEQ ID NO: 669), SGGTLAS (SEQ ID NO: 670), LGGTLAS (SEQ ID NO: 671), TGGTLAS (SEQ ID NO: 672), GGGTLAS (SEQ ID NO: 673), RGGNLAS (SEQ ID NO: 674), DGGDLAS (SEQ ID NO: 675), DGGKLAS (SEQ ID NO: 676), QGGKLAS (SEQ ID NO: 677), RGGKLAS (SEQ ID NO: 678), LGGKLAS (SEQ ID NO: 679), SGGKLAS (SEQ ID NO: 680), GGGKLAS (SEQ ID NO: 681), DGGRLAS (SEQ ID NO: 682), DGGNRAT (SEQ ID NO: 683), RGGTLES (SEQ ID NO: 684), KGGTLES (SEQ ID
  • the antibody variable domains of the present invention exhibit a reduced predicted binding to MHC class II proteins, more specifically to human MHC class II molecules, when compared to their original versions that do not comprise said GG motif.
  • the NetMHCIIpan algorithm was used, more specifically version 3.1 of the NetMHCIIpan software and NetMHCIIpan algorithm, respectively, hereinafter referred to as “NetMHCIIpan-3.1” or “NetMHCIIpan-3.1 algorithm”, as described in detail in Example 2.
  • the immunogenicity risk of a given (therapeutic) protein is determined from the predicted binding strength of all 15mer peptide segments within the (therapeutic) protein to MHC class II molecules. These binding strengths are expressed by percentile ranks, which can be plotted over the analyzed sequence as for example shown in Figure 1. The lower the percentile rank, the stronger is the predicted binding strength to MHC class II molecules.
  • variable light chain and/or the variable heavy chain, in particular the variable light chain, of the antibody variable domain of the present invention comprises no 15mer peptide stretch having a median percentile rank, as determined from the predicted individual binding strengths (scoring ranks) of said 15mer peptide to each one of the 27 MHC class II alleles that are most common in the general population using the algorithm of NetMHCIIpan, of less than 13, particularly less than 14, particularly less than 15, particularly less than 16, particularly less than 17, particularly less than 18, particularly less than 19, particularly less than 20.
  • the NetMHCIIpan-3.1 algorithm allows a reliable in silico prediction of immunogenicity risk of an antibody or antibody fragments.
  • the term “immunogenicity” or “immunogenicity risk”, as used herein, refers to the capacity of a therapeutic protein, e. g. an antibody, an antibody fragment or an antibody variable domain, to bind to MHC class II proteins.
  • a therapeutic protein e. g. an antibody, an antibody fragment or an antibody variable domain
  • the induction of the formation of ADAs during therapeutic treatment is linked with the occurrence of B cell and/or T cell epitopes on a therapeutic protein.
  • the extent of such immunogenicity can also be estimated by an ELISA assay and can be expressed by the percentage or the number of human serum samples, which contain measurable amounts of ADAs formed during therapeutic treatment, that recognize, i. e. bind to, the therapeutic protein in question, relative to the total number of tested human sera (percentage or number of positive serum samples).
  • a reduction of immunogenicity between a therapeutic protein and a corresponding therapeutic protein being modified with the goal to reduce its immunogenicity e. g. by implementing the GG motif described herein, can be measured by comparing the percentage of positive serum samples against the modified therapeutic protein, with the percentage of positive serum samples against the original therapeutic protein.
  • the antibody variable domains of the present invention when being in scFv format, further have advantageous biophysical properties, in particular an excellent stability.
  • the antibody variable domains of the present invention maintain their stability, as indicated by their melting temperature (Tm), when compared to their original versions that do not comprise the GG motif.
  • Tm melting temperature
  • the expression “maintain their stability” means that their Tm is either equal or lower than the Tm of their respective unmodified versions (i. e.
  • the expression “maintain their binding affinity” means that the monovalent dissociation constant (KD) for binding of the antibody variable domains of the invention to their target antigens, as measured by surface plasmon resonance (SPR), is either equal or lower than the KD of their respective unmodified versions (i. e. versions of said antibody variable domains that do not comprise the LCDR2 amino acids defined in item 1), or is not more than 2 times greater than the KD of their original versions (i. e. versions of said antibody variable domains that do not comprise the LCDR2 amino acids defined in item 1).
  • SPR surface plasmon resonance
  • the antibody variable domain of the present invention when being in scFv format, is further characterized by one or more of the following features: (i) has a melting temperature (Tm), when compared with the Tm of a version of said antibody variable domain that does not comprise the LCDR2 amino acids defined in item 1, that is not lower than 5°C, particularly not lower than 4.5°C, particularly not lower than 4°C, particularly not lower than 3.5°C, particularly not lower than 3.0°C, as determined by differential scanning fluorimetry (DSF) and when formulated in 20 mM Histidine, pH 6.0; (ii) binds its target antigen with a dissociation constant (KD) that is not greater than 2-folds, particularly not greater than 1.7-folds, particularly not greater than 1.5-folds, particularly not greater than 1.4-folds, particularly not greater than 1.3-folds, particularly not greater than 1.2-folds, when compared with the KD of a version of said antibody variable 119523P877
  • DSF is described earlier (Egan, et al., MAbs, 9(1) (2017), 68-84; Niesen, et al., Nature Protocols, 2(9) (2007) 2212-2221).
  • the midpoint of transition for the thermal unfolding of the scFv constructs is determined by nano Differential Scanning Fluorimetry as described in detail in Example 4.
  • 1 ⁇ 0.1 mg/ml and 10 ⁇ 1 mg/ml solution in 20 mM Histidine buffer, pH 6, are prepared and subjected to a temperature ramp of from 20°C to 95°C with a 1°C/min increase.
  • the unfolding event is monitored by using the intrinsic fluorescence of proteins, i. e.
  • the term “affinity” refers to the strength of interaction between the antibody or the antibody variable domain and the antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody variable domain or the antibody “arm” interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity. [0082] “Binding affinity” generally refers to the strength of the total sum of non-covalent interactions between a single binding site of a molecule (e.
  • binding affinity refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e. g., an antibody variable domain and an antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.
  • binding affinity i. e. binding strength
  • Kassoc Ka or “Kon”, as used herein, are intended to refer to the association rate of a particular antibody-antigen interaction
  • Kdis Kd or “Koff”, as used herein, is intended to refer to the dissociation rate of a particular antibody- antigen interaction.
  • KD is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i. e. Kd/Ka) and is 119523P877PC 17.05.2024 Numab Therapeutics AG expressed as a molar concentration (M).
  • M molar concentration
  • the “KD” or “KD value” or “KD” or “KD value” according to this invention is in one embodiment measured by using surface plasmon resonance assays.
  • the antibody variable domains of the present invention may have further modifications that can reduce immunogenicity.
  • the antibody variable domains of the present invention additionally comprise substitutions in the framework regions to reduce their potential binding to pre-existing ADAs, when being in an antibody fragment-based format, such as in an Fv or scFv format. Examples of such modifications are for example disclosed in the patent applications WO 2022/136693 and WO 2023/214047.
  • the antibody variable domains of the present invention comprise a variable heavy chain (VH) having a serine (S) at amino acid position 101 and a lysine (K) at amino acid position 146 (AHo numbering.
  • the present invention relates to an antibody comprising one or more antibody variable domains of the present invention.
  • the antibody of the present invention further comprises antibody variable domains that differ from the antibody variable domains of the present invention. More specifically, the antibody of the present invention further comprises antibody variable domains that do not have the GG motif as defined herein.
  • the antibody of the present invention exclusively comprises antibody variable domains of the present invention.
  • the antibody of the present invention exclusively comprises antibody variable domains that have the substitutions in the framework regions as defined herein.
  • binding domain or “monovalent binding domain”, as used herein, refers to a binding domain that binds to a single epitope on a target molecule.
  • binding domain of an antibody, as used herein, or the terms “antigen-binding fragment thereof” or “antigen-binding portion” of an antibody, and the like, refer to one or more parts of an intact antibody that have the ability to bind to a given antigen, in particular to specifically bind to a given antigen.
  • Antigen-binding functions of an antibody can be performed by fragments of an intact antibody.
  • binding domain as used herein, or the terms 119523P877PC 17.05.2024 Numab Therapeutics AG “antigen-binding fragment thereof” or “antigen-binding portion”, and the like, refer to a Fab fragment, i. e.
  • the binding domains of the antibodies of the present invention are independently of each other selected from a Fab fragment, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) and a single- chain Fv fragment (scFv).
  • the binding domains of the antibodies of the present invention are independently of each other selected from a Fab fragment an Fv fragment and a single-chain Fv fragment (scFv).
  • the VL and VH domains of the scFv fragment are stabilized by an interdomain disulfide bond, in particular said VH domain comprises a single cysteine residue in position 51 (AHo numbering) and said VL domain comprises a single cysteine residue in position 141 (AHo numbering).
  • bivalent antibody or “antibody that is bivalent for its target antigen”, as used herein, refers to a single antibody with two valencies, where “valency” is described as the number of antigen-binding moieties that binds to epitopes on a specific target molecule.
  • the single antibody can bind to two binding sites on a target molecule and/or to two target molecules due to the presence of two copies of the corresponding antigen-binding moieties.
  • the term “trivalent antibody” or “antibody that is trivalent for its target antigen”, as used herein, refers to a single antibody with three valencies.
  • the single antibody can bind to three binding sites on a target molecule and/or can bind up to three target molecules due to the presence of three copies of the corresponding antigen-binding moieties.
  • the antibodies of the invention comprise two or three binding domains, said two or three binding domains either bind the same epitope or different epitopes on the target molecules.
  • the two or three binding domains bind the same epitope on the target molecule.
  • the term “same epitope”, as used herein, refers to an individual protein determinant on the protein capable of specific binding to more than one antibody, where that individual protein determinant is identical, i. e. consist of identical chemically active surface groupings of molecules such as amino acids or sugar side chains having identical three-dimensional structural characteristics, as well as identical charge characteristics for each of said antibodies.
  • the format of the antibody is selected from KiH-based IgGs, such as DuoBodies (bispecific IgGs prepared by the Duobody technology) (MAbs. 2017 Feb/Mar;9(2):182-212.
  • DVD-Ig IgG-scFv fusions, such as CODV-IgG, Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)), bsAb (scFv linked to C-terminus of light chain), Bs1Ab (scFv linked to N-terminus of light chain), Bs2Ab (scFv linked to N-terminus of heavy chain), Bs3Ab (scFv linked to C-terminus of heavy chain), Ts1Ab (scFv linked to N-terminus of both heavy chain and light chain) and Ts2Ab (dsscFv linked to C-terminus of heavy chain).
  • IgG-scFv fusions such as CODV-IgG, Morrison (IgG CH3-scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)
  • the format of said antibody is selected from KiH-based IgGs, such as DuoBodies; DVD-Ig; CODV-IgG and Morrison (IgG CH 3 -scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)), even more particularly from DVD-Ig and Morrison (IgG CH 3 -scFv fusion (Morrison-H) or IgG CL-scFv fusion (Morrison-L)).
  • the IgG is preferably selected from the IgG subclasses IgG1 and IgG4, in particular IgG4.
  • the format of said antibody is selected from a Morrison format, i. e. a Morrison-L and a Morrison-H format.
  • the Morrison-L and Morrison-H format used in the present invention are tetravalent and bispecific molecular formats bearing an IgG Fc region, in particular an IgG4 Fc region.
  • Two highly stable scFv binding domains, wherein the light chain comprises V ⁇ FR1 to FR3 in combination with a V ⁇ FR4, and which are based on the antibody variable domains of the present invention, are fused via a linker L1 to the heavy chain (Morrison-H) or light chain (Morrison-L) C-termini.
  • the linker L1 is a peptide of 2-30 amino acids, more particularly 5-25 amino acids, and most particularly 10-20 amino acids.
  • GGGGS serine amino acid residue
  • the VH regions and the VL regions of the two scFv domains are connected by a linker L2.
  • the linker L2 is a peptide of 10-40 amino acids, more particularly 15-30 amino acids, and most particularly 20-25 amino acids.
  • the antibody of the invention does not comprise an immunoglobulin Fc region.
  • the term “immunoglobulin Fc region” or “Fc region”, as used herein, is used to define a C-terminal region of an immunoglobulin heavy chain, i. e. the CH2 and CH3 domains of the heavy chain constant regions.
  • Fc region includes native-sequence Fc regions and variant Fc regions, i. e. Fc regions that are engineered to exhibit certain desired properties, such as for example altered Fc receptor binding function and/or reduced or suppressed Fab arm exchange.
  • An example of such an engineered Fc region is the knob- into-hole (KiH) technology (see for example Ridgway et al., Protein Eng.9:617-21 (1996) and Spiess et al., J Biol Chem.288(37):26583-93 (2013)).
  • Native-sequence Fc regions include human lgG1, lgG2 (lgG2A, IgG2B), lgG3 and lgG4.
  • Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
  • the FcR is a native sequence human FcR, which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, FcyRII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors, Fc ⁇ RII receptors including Fc ⁇ RIIA (an "activating receptor”) and Fc ⁇ RI IB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see M. Daeron, Annu. Rev. Immunol. 5:203-234 (1997).
  • FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol.9: 457-92 (1991); Capet et al, Immunomethods 4: 25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126: 330-41 (1995).
  • FcR Fc receptor
  • FcRn neonatal receptor
  • Binding to FcRn in vivo and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, e. g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides having a variant Fc region are administered.
  • WO 2004/42072 (Presta) describes antibody variants which improved or diminished binding to FcRs.
  • the antibody is preferably in a format selected from the group consisting of: a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a circular dimeric scDb (CD-scDb), a tandem tri-scFv, a tribody (Fab-(scFv)2), a Fab-Fv2, a triabody, an scDb-scFv, a tetrabody, a di-diabody, a tandem-di-scFv and a MATCH (described in WO 2016/0202457; Egan T., et al., MABS 9 (2017) 68-84).
  • the antibody of the invention does further not comprise CH1 and/or CL regions.
  • the antibody is in a scDb-scFv, a triabody, a tetrabody or a MATCH format, particularly in a MATCH or scDb-scFv format. More particularly, the antibody of the invention is in a MATCH3 or a MATCH4 format.
  • the antibody of the invention is trispecific and tetravalent.
  • the antibody of the invention is trispecific and trivalent.
  • the antibody variable domains comprised in the bispecific, trispecific tetraspecific or pentaspecific, antibodies of the invention are capable of binding to their respective antigens or receptors simultaneously.
  • the term “simultaneously”, as used in this connection refers to the simultaneous binding of one of the antibody variable domains, which for example specifically binds to PD-L1, and of one or two further antibody variable domains, which for example have specificity for CD137 and hSA.
  • the antibody variable domains comprised in the bispecific, trispecific tetraspecific or pentaspecific, antibodies of the invention are operably linked.
  • the term “operably linked”, as used herein, indicates that two molecules (e.
  • polypeptides, domains, binding domains are attached in a way that each molecule retains functional activity.
  • Two molecules can be “operably linked” whether they are attached directly or indirectly (e. g., via a linker, via a moiety, via a linker to a moiety).
  • linker refers to a peptide or other moiety that is optionally located between binding domains or antibody variable domains used in the invention.
  • a number of strategies may be used to covalently link molecules together. These include, but are not limited to, polypeptide linkages between N- and C-termini of proteins or protein domains, linkage via disulfide bonds, and linkage via chemical cross-linking reagents.
  • the linker is a peptide bond, generated by recombinant techniques or peptide synthesis. Choosing a suitable linker for a specific case where two polypeptide chains are to be connected depends on various parameters, including but not limited to the nature of the two polypeptide chains (e. g., whether they naturally oligomerize), the distance between the N- and the C-termini to be connected if known, and/or the stability of the linker towards proteolysis and oxidation. Furthermore, the linker may contain amino acid residues that provide flexibility.
  • polypeptide linker refers to a linker consisting of a chain of amino acid residues linked by peptide bonds that is connecting two domains, each being attached to one end of the linker.
  • the polypeptide linker should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity.
  • the polypeptide linker has a continuous chain of between 2 and 30 amino acid residues (e. g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues).
  • the amino acid residues selected for inclusion in the polypeptide linker should exhibit properties that do not interfere significantly with the activity of the polypeptide.
  • the linker peptide on the whole should not exhibit a charge that would be inconsistent with the activity of the polypeptide, or interfere with internal folding, or form bonds or other interactions with amino acid residues in one or more of the monomers that would seriously impede the binding of receptor monomer domains.
  • the polypeptide linker is non-structured polypeptide.
  • Useful linkers include glycine-serine, or GS linkers.
  • Gly-Ser or “GS” linkers is meant a polymer of glycines and serines in series (including, for example, (Gly-Ser) n , (GSGGS) n (SEQ ID NO: 541), (GGGGS) n (SEQ ID NO: 542) and (GGGS) n (SEQ ID NO: 543), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers such as the tether for the shaker potassium channel, and a large variety of other flexible linkers, as will be appreciated by those in the art.
  • the antibody variable domain of the invention is an isolated variable domain.
  • the antibodies of the invention are isolated antibodies.
  • isolated variable domain refers to a variable domain or an antibody that is substantially free of other variable domains or other antibodies having different antigenic specificities (e. g., an isolated antibody variable domain that specifically binds mesothelin is substantially free of antibody variable domains that specifically bind antigens other than mesothelin). Moreover, an isolated antibody variable domain or isolated antibody may be substantially free of other cellular material and/or chemicals. [0112] Suitably, the antibody variable domains and antibodies of the invention are monoclonal antibody variable domains and antibodies.
  • variable domains or “monoclonal antibody” as used herein refers to variable domains or 119523P877PC 17.05.2024 Numab Therapeutics AG antibodies that have substantially identical amino acid sequences or are derived from the same genetic source.
  • a monoclonal variable domain or antibody displays a binding specificity and affinity for a particular epitope, or binding specificities and affinities for specific epitopes.
  • the antibody variable domains and antibodies of the invention include, but are not limited to, chimeric, human and humanized antibody variable domains and antibodies.
  • chimeric antibody or “chimeric antibody variable domain”, as used herein, refers to an antibody molecule or antibody variable domain in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • a mouse antibody can be modified by replacing its constant region with the constant region from a human immunoglobulin.
  • human antibody or “human antibody variable domain” , as used herein, is intended to include antibodies or antibody variable domains having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody or antibody variable domain contains a constant region, the constant region also is derived from such human sequences, e. g., human germline sequences, or mutated versions of human germline sequences.
  • the human antibodies and antibody variable domains of the invention may include amino acid residues not encoded by human sequences (e.
  • Human antibodies and antibody variable domains specifically excludes a humanized antibody or antibody variable domain comprising non-human antigen-binding residues.
  • Human antibodies and antibody variable domains can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1992); Marks et al, J. Mol. Biol, 222:581 (1991)). Also available for the preparation of human monoclonal antibodies and human monoclonal antibody variable domains are methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Human antibodies and human antibody variable domains can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies and antibody variable domains in response to antigenic challenge, but whose 119523P877PC 17.05.2024 Numab Therapeutics AG endogenous loci have been disabled, e. g., immunized xenomice (see, e. g., U.S. Pat. Nos.
  • humanized antibody or “humanized” antibody variable domain refers to an antibody or antibody variable domain that retains the reactivity of a non- human antibody or antibody variable domain while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody or antibody variable domain with their human counterparts (i. e. the constant region as well as the framework portions of the variable region).
  • humanized antibodies and antibody variable domains of the invention may include amino acid residues not encoded by human sequences (e. g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing). See, e. g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv.
  • the antibody variable domains and antibodies of the invention are humanized.
  • the antibody variable domains and antibodies of the invention are humanized and comprise rabbit-derived CDRs.
  • the antibodies of the invention are bispecific, trispecific or tetraspecific, particularly bispecific or trispecific, more particularly trispecific.
  • trispecific antibody refers to an antibody that binds to at least three different epitopes on three different targets (e. g., PD-L1, CD137 and hSA or mesothelin, CD3 and hSA).
  • targets e. g., PD-L1, CD137 and hSA or mesothelin, CD3 and hSA.
  • epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. “Conformational” and “linear” epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • the term “conformational epitope” as used herein refers to amino acid residues of an antigen that come together on the surface when the polypeptide chain folds to form the native protein.
  • the term “linear epitope” refers to an epitope, wherein all points of interaction between the protein and the interacting molecule (such as an antibody) occurring linearly along the primary amino acid sequence of the protein (continuous).
  • the term “recognize” as used herein refers to an antibody or antibody variable domain that finds and interacts (e. g., binds) with its conformational epitope.
  • the inventors found that the antibody variable domain of the invention can be successfully applied in the construction of diverse antibody fragments, e. g.
  • the antibody variable domains and antibodies of the invention can be produced using any convenient antibody-manufacturing method known in the art (see, e. g., Fischer, N. & Leger, O., Pathobiology 74 (2007) 3-14 with regard to the production of bispecific constructs; Hornig, N. & Desirber-Schwarz, A., Methods Mol. Biol.907 (2012)713-727, and WO 99/57150 with regard to bispecific diabodies and tandem scFvs).
  • suitable methods for the preparation of the multispecific constructs further include, inter alia, the Genmab (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150) and Merus (see de Kruif et al., Biotechnol. Bioeng.106 (2010) 741-750) technologies.
  • Genmab see Labrijn et al., Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150
  • Merus see de Kruif et al., Biotechnol. Bioeng.106 (2010) 741-750
  • These methods typically involve the generation of monoclonal antibodies or monoclonal antibody variable domains, for example by means of fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen using the hybridoma technology (see, e. g., Yokoyama et al., Curr. Protoc. Immunol.
  • bispecific, trispecific, tetraspecific or pentaspecific, and/or multivalent can be prepared by conjugating the constituent binding specificities, using methods known in the art. For example, each binding specificity of these antibodies can be generated separately and then conjugated to one another.
  • the binding specificities are proteins or peptides
  • a variety of coupling or cross-linking agents can be used for covalent conjugation.
  • the bispecific molecule is a mAb x Fab, a mAb x scFv, a mAb x dsFv or a mAb x Fv fusion protein.
  • Methods for preparing multispecific and/or multivalent antibodies and molecules are described for example in US 5,260,203; US 5,455,030; US 4,881,175; US 5,132,405; US 5,091,513; US 5,476,786; US 5,013,653; US 5,258,498; and US 5,482,858.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphorates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e. g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081, 1991; Ohtsuka et al., J. Biol. Chem.260:2605- 2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).
  • the invention provides substantially purified nucleic acid molecules which encode polypeptides comprising segments or domains of the antibody variable domains or the antibodies described above.
  • polypeptides encoded by these nucleic acid molecules are capable of exhibiting antigen- binding capacities of the antibody variable domains or the antibodies of the present invention.
  • the polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e. g., sequences as described in the Examples below) encoding the antibody variable domain or the antibody of the invention.
  • Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol.68:90; the phosphodiester method of Brown et al., Meth.
  • vectors and host cells for producing the antibody variable domain or the antibody of the invention. 119523P877PC 17.05.2024 Numab Therapeutics AG [0135]
  • vector is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e. g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e. g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. [0136] Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e. g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • viral vectors e. g., replication defective retroviruses, adenoviruses and adeno- associated viruses
  • the term “operably linked” refers to a functional relationship between two or more polynucleotide (e. g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i. e. they are cis-acting.
  • some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • Various expression vectors can be employed to express the polynucleotides encoding the antibody variable domain or the antibody chain(s).
  • Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e. g., Harrington et al., Nat Genet.15:345, 1997).
  • g., human cells include pThioHis A, B and C, pcDNA3.1/His, pEBVHis A, B and C, (Invitrogen, 119523P877PC 17.05.2024 Numab Therapeutics AG San Diego, Calif.), MPS V vectors, and numerous other vectors known in the art for expressing other proteins.
  • Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, Vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev.
  • the choice of expression vector depends on the intended host cells in which the vector is to be expressed.
  • the expression vectors contain a promoter and other regulatory sequences (e. g., enhancers) that are operably linked to the polynucleotides encoding a multispecific antibody chain or a variable domain.
  • an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions.
  • Inducible promoters include, e. g., arabinose, lacZ, metallothionein promoter or a heat shock promoter.
  • Cultures of transformed organisms can be expanded under non-inducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells.
  • other regulatory elements may also be required or desired for efficient expression of a multispecific antibody chain or a variable domain. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences.
  • the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e. g., Scharf et al., Results Probl. Cell Differ.20: 125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987).
  • the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.
  • Vectors to be used typically encode the antibody variable domain or the antibody light and heavy chain including constant regions or parts thereof, if present. Such vectors allow expression of the variable regions as fusion proteins with the constant regions thereby leading to production of intact antibodies and antibody variable domains thereof. Typically, such constant regions are human.
  • the term “recombinant host cell” (or simply “host cell”) refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell.
  • the host cells for harboring and expressing the antibody variable domain or the antibody of the invention can be either prokaryotic or eukaryotic.
  • E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present invention.
  • microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • prokaryotic hosts one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e. g., an origin of replication).
  • expression vectors typically contain expression control sequences compatible with the host cell (e. g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • Other microbes such as yeast, can also be employed to express the antibody variable domain or multispecific antibodies of the invention.
  • Insect cells in combination with baculovirus vectors can also be used.
  • mammalian host cells are used to express and produce the antibody variable domain or the antibody of the invention.
  • they can be either a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell line harboring an exogenous expression vector. These include any normal mortal or normal or abnormal immortal animal or human cell.
  • a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed including the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas.
  • the use of mammalian tissue cell culture to express polypeptides is discussed generally in, e. g., Winnacker, FROM GENES TO CLONES, VCH Publishers, N.Y., N.Y., 1987.
  • Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e. g., Queen, et al., Immunol.
  • expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable.
  • Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art. [0143] Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host.
  • calcium chloride transfection is commonly utilized for prokaryotic cells
  • calcium phosphate 119523P877PC 17.05.2024 Numab Therapeutics AG treatment or electroporation may be used for other cellular hosts.
  • Other methods include, e.
  • cell lines which stably express the antibody variable domain or the antibody of the invention can be prepared using expression vectors of the invention which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1 to 2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media.
  • Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • the present invention thus provides a method of producing the antibody variable domain or the antibody of the invention, wherein said method comprises the step of culturing a host cell comprising a nucleic acid or a vector encoding the antibody variable domain or the antibody of the invention, whereby said antibody variable domain or said antibody of the disclosure is expressed.
  • the present invention relates to a method of producing the antibody variable domain or the antibody of the invention, the method comprising the step of culturing a host cell expressing a nucleic acid or two nucleic acids encoding the antibody variable domain or the antibody of the invention.
  • the present invention relates to a method of producing the antibody variable domain or the antibody of the invention, the method comprising (i) providing a nucleic acid or two nucleic acids encoding the antibody variable domain or the antibody of the invention or one or two vectors encoding the antibody variable domain or the antibody of the invention, expressing said nucleic acid or nucleic acids, or said vector or vectors, and collecting said antibody variable domain or said antibody from the expression system, or (ii) providing a host cell or host cells expressing a nucleic acid or two nucleic acids encoding the antibody variable domain or the antibody of the invention, culturing said host cell or said host cells; and collecting said antibody variable domain or said multispecific antibody from the cell culture.
  • compositions in accordance with the present disclosure may further routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.
  • the composition may also include antioxidants and/or preservatives.
  • antioxidants may be mentioned thiol derivatives (e. g. thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol, glutathione), tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, sulfurous acid salts (e. g. sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) and nordihydroguaiaretic acid.
  • thiol derivatives e. g. thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol, glutathione
  • tocopherols e. g. thiogly
  • the present invention relates to a method of treating a subject comprising administering to the subject a therapeutically effective amount of the antibody of the present invention.
  • the present invention relates to a method for the treatment of a proliferative disease, such as cancer, or a disease selected from allergic, inflammatory and autoimmune diseases, in a subject comprising administering to the subject a therapeutically effective amount of the antibody of the present invention.
  • a proliferative disease such as cancer
  • a disease selected from allergic, inflammatory and autoimmune diseases in a subject comprising administering to the subject a therapeutically effective amount of the antibody of the present invention.
  • the term “subject” includes human and non-human animals.
  • the term “animals” include all vertebrates, e.
  • terapéuticaally effective amount refers to the amount of an agent that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the agent, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • Example 1 Manufacturing of GG variants of scFvs and multispecific antibodies according to the present invention and unmodified versions thereof (References) 1.1. Manufacturing of GG variants of scFvs according to the present invention and unmodified variants thereof (References): [0169] The scFv GG variants according to the present invention as well as their respective unmodified versions (non-GG variants) have been produced following the methods described in detail in the patent applications WO 2019/072868 and WO 2021/239987. The produced scFv GG variants and corresponding non-GG versions are listed in Table 5.
  • scFv GG variants as defined herein, and their respective unmodified versions (References) was performed in CHO cells using the ExpiCHO Expression System (ThermoFisher). Expression was conducted according to manufacturer’s instructions. Proteins were purified from clarified harvest by affinity chromatography (Protein L and/or Protein A). If necessary, variant scFvs were polished by SE-chromatography to a final monomeric content > 95 %. For quality control of the manufactured material, standard analytical methods such as ESI-MS, SE-HPLC, UV280 and SDS-PAGE were applied. [0171] The mass of the scFvs has for example been verified by the following ESI-MS standard method.
  • scFvs were 5-fold diluted with 1% TFA.
  • Two ⁇ l of sample were injected into an ACQUITY UPLC@ BioResolve-RP-mAb 2.7 ⁇ m 2.1x150 mm, 450 ⁇ column (Waters, USA) and desalted using a gradient from 15 % to 85 % buffer B (0.1 % formic acid, 25 % propan-2-ol in acetonitrile) at a flow rate of 200 ⁇ l/min at 50°C.
  • the MS analysis was performed on a Synapt G2 mass spectrometer directly coupled to the UPLC station.
  • Mass spectra were acquired in the positive-ion mode by scanning the m/z range from 400 to 5,000 da with a scan duration of 1 s and an interscan delay of 0.1s.
  • the data were recorded with the MassLynx 4.2 Software (both Waters, UK). Where possible, the recorded m/z data of single peaks were deconvoluted into mass spectra by applying the maximum entropy algorithm MaxEnt1 (MaxLynx).
  • MaxEnt1 MaxLynx
  • PRO1480 anti-PDL1 x CD137 x hSA multispecific antibody
  • GG variants of PRO1480 [0174] The characterization and manufacturing of PRO1480 and its binding domains is disclosed in detail in the patent application WO 2019/072868.
  • the PRO1480 GG variants of the present invention and its non-modified versions (References) have been produced according to the methods described therein.
  • the produced PRO1480 GG variants and corresponding non-GG versions are listed in Table 5. [0175] Briefly, the expression of PRO1480 GG variants and non-modified versions thereof (scMATCH3 constructs) has been performed at 0.5 l scale using CHOgro expression kit (Mirus) and mammalian CHO-S cells.
  • proteins were purified from clarified culture supernatants by Protein A (MabSelect PrismA, Cytiva) affinity chromatography either followed by size exclusion chromatography (SEC) in 50 mM phosphate-citrate buffer with 300 mM sucrose at pH 6.5 or, where applicable, capture fractions with >95 % purity were directly pooled and buffer exchanged to 50 mM phosphate- citrate buffer with 300 mM sucrose at pH 6.5 buffer. Monomeric content of SEC fractions was assessed by SE-HPLC analysis and fractions with a monomeric content >95 % were pooled. For quality control of the manufactured material, standard analytical methods such as SE- HPLC, UV280 and SDS-PAGE were applied.
  • SEC size exclusion chromatography
  • NetMHCIIpan operates by splitting each amino acid sequence into consecutive overlapping fixed-length frames.
  • NetMHCIIpan uses 15mer frames, and within each such frame, the lowest scoring 9mer is identified and reported as the score of the 15mer, for each chosen allele. For the prediction results reported herein, a set of 27 alleles was used, which covers a substantial majority of MHC class II molecules (Greenbaum et al., Immunogenetics, 2011, 63, pp.325-335). The per-allele percentile rank of a 15mer is obtained from its score by comparing the score against the scores of 200,000 random natural 15mers (Karosiene et al., Immunogenetics, 2013, 65, pp.711-724).
  • a median percentile rank less than 20 is the commonly chosen threshold for this purpose (Oseroff et al., J. Immunol., 2010, 185, pp.943- 955; Paul et al., J. Immunol. Methods, 2015, 422, pp.28-34).
  • the NetMHCIIpan software predicts binding of each individual peptide to MHC class II by applying an ensemble of artificial neural networks with 906-neuron input layers, that have been trained on quantitative peptide binding data that covers multiple MHC class II molecules.
  • the amino 119523P877PC 17.05.2024 Numab Therapeutics AG acids of each 9mer core peptide are encoded as the corresponding 20-element vector from the BLOSUM50 matrix (Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, pp.10915- 10919).
  • the peptide flanking regions are encoded as one 20-element vector for each side of the binding core, formed as the average BLOSUM50 score vector for the residues in each flanking region.
  • MHC class II molecules are represented by 34-residue pseudo-sequences, where each residue is again encoded as the corresponding 20-element vector from the BLOSUM50 matrix.
  • the aim of these thermal stability measurement was to determine the influence of the GG modification on the stability of the respective scFvs.
  • the unfolding event causes a shift of the fluorescence emission spectra of tryptophan (Trp) by a change of its environment, e. g., from the hydrophobic core to solvent-exposed region.
  • Tm thermal midpoint
  • Tm is an important factor to assess the stability of protein. Higher Tm suggests a more stable protein.
  • a protein can have multiple Tm based on the number of domains that unfold separately.
  • the thermal stability is further influenced by the protein concentration and buffer conditions.
  • the following general procedure was applied. Proteins in 20 mM histidine buffer (pH 6) at 1 ⁇ 0.1 mg/ml were prepared for thermal stability. Melting curves were generated with a temperature increase from 20°C to 95°C with a 1°C/min. The intrinsic fluorescence of the proteins was monitored and recorded. The spectral shift upon unfolding was recorded at two wavelengths, 330 nm and 350 nm, and the ratio of 350 nm/330 nm was taken to analyze the data.
  • the protein unfolding was conducted using the Prometheus device (Nanotemper Technologies) and analyzed by the “PR.ThermControl” v2.3.1 and “PR.Stability Analysis” v1.1 software.
  • the Tm values obtained for the GG modified scFvs variants do not vary much from the Tm values obtained for the corresponding reference compounds. Almost all GG variants exhibit high Tm values indicating excellent stability also for the GG modified scFvs variants.
  • These results indicates that the GG modification (67G and 68G) in the CDRL2 of the tested scFvs does not significantly influence their thermal stability. .

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Abstract

La présente invention concerne des domaines variables d'anticorps, présentant une liaison réduite aux anticorps anti-médicaments (ADA), des anticorps contenant un ou plusieurs de ces domaines variables d'anticorps, et des compositions pharmaceutiques contenant ces anticorps. La présente invention concerne également des acides nucléiques codant pour lesdits domaines variables d'anticorps ou lesdits anticorps, un ou plusieurs vecteurs comprenant lesdits acides nucléiques, une ou plusieurs cellules hôtes comprenant lesdits acides nucléiques ou ledit/lesdits vecteur(s) ainsi qu'un procédé de production desdits domaines variables d'anticorps ou lesdits anticorps multispécifiques. De plus, la présente invention concerne un procédé de production desdits domaines variables d'anticorps et des anticorps.
PCT/EP2024/063788 2023-05-19 2024-05-17 Domaines variables d'anticorps et anticorps présentant une immunogénicité réduite Pending WO2024240690A2 (fr)

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