US20210047395A1 - Method for improving vegf-receptor blocking selectivity of an anti-vegf antibody - Google Patents
Method for improving vegf-receptor blocking selectivity of an anti-vegf antibody Download PDFInfo
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- US20210047395A1 US20210047395A1 US16/912,877 US202016912877A US2021047395A1 US 20210047395 A1 US20210047395 A1 US 20210047395A1 US 202016912877 A US202016912877 A US 202016912877A US 2021047395 A1 US2021047395 A1 US 2021047395A1
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C07K2317/565—Complementarity determining region [CDR]
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/567—Framework region [FR]
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- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Definitions
- the present invention relates to methods for improving anti-VEGF antibodies in order to provide or improve antibodies that preferentially inhibit binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1; and antibodies provided by said methods.
- Anti-VEGF antibodies that are approved for clinical application, such as Avastin® and Lucentis®, inhibit VEGF-binding to both receptors, VEGF-R1 (FLT-1, fms-like tyrosine kinase) and VEGF-R2 (KDR/FLK-1, fetal liver kinase).
- VEGF-R1 and VEGF-R2 are closely related receptor tyrosine kinases (RTK). While VEGF-R2 is hypothized to be primarily responsible for VEGF-mediated angiogenesis (Holash, J. et al., Proc Natl Acad Sci USA. 2002 Aug.
- VEGF-R1 is known to have other important biological roles unrelated to angiogenesis, e.g. in osteoclast differentiation (Aldridge, S. E. et al., Biochem Biophys Res Commun. 2005 Sep. 30; 335(3):793-8).
- the present invention relates to a method of improving VEGFR-blocking selectivity of an antibody that binds to VEGF comprising an antigen binding site formed by cognate pair of a VH and a VL domain, wherein the antibody binds to an epitope of VEGF that overlaps with the VEGF-R1-binding region and the VEGF-R2-binding region in the VEGF molecule.
- the method of the invention comprises (a) providing an analysis of the tertiary structure of a complex of a VEGF-dimer bound by a first and a second antigen binding site of said antibody that binds to VEGF (VEGF-dimer-antibody-complex), (b) identifying at least one amino acid residue located in the VH domain or VL domain of said antibody, wherein said amino acid residue within the first antigen binding site and said amino acid residue within the second antigen binding site are spatially arranged in close proximity in the VEGF-dimer-antigen-complex; and (c) substituting said at least one amino acid residue identified in step b) by an amino acid having a larger side chain volume.
- VEGFR-blocking selectivity of certain anti-VEGF antibodies may be improved by a few modifications in their amino acid sequence, particularly in regions that are not involved in antigen binding.
- Such antibodies may exhibit an improved safety profile, e.g. by avoiding or reducing side effects caused by blocking VEGF-signalling through VEGF-R1.
- the antibody binds to a conformational epitope on a dimer of VEGF-A121, wherein VEGF-A121 comprises an amino acid sequence of SEQ ID NO: 36, wherein the epitope comprises in one of the individual VEGF-A121 molecules within the VEGF dimer amino acids F17, M18, D19, Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, L66, N100, K101, C102, E103, C104, R105 and P106; and in the other one of the individual VEGF-A121 molecules within the VEGF dimer amino acids E30, K48, M81 and Q87.
- the epitope is measured by x-ray crystallography.
- the amino acid with a larger side chain volume is an aromatic amino acid.
- the at least one substituted amino acid residue is located in the heavy chain variable domain of said antibody.
- Another aspect of the invention is an antibody that binds to VEGF provided by a method of one of the invention.
- Another aspect of the invention is antibody that binds to VEGF, wherein binding of the antibody to VEGF significantly inhibits VEGF-binding to VEGF receptor VEGF-R2 without significantly inhibiting VEGF-binding to VEGF receptor VEGF-R1, provided by a method of the invention.
- FIG. 1 Crystal structure of VEGF dimer (purple) in complex with VEGF-R1 domain 2 (red) and with VEGF-R2 domain 2 and 3 (blue)
- FIG. 2 Inhibition of VEGF-binding to VEGF-R1 and VEGF-R2 in presence of antibody Fab fragments (VEGF:VEGF-R2/R1 inhibitition ELISA) as described in Example 2.
- FIG. 3 Crystal structure of VEGF dimer (purple) in complex with anti-VEGF antibody VEGF-0089 as determined by X ray crystallography according to Example 3. Red circle highlights regions in the VH domain of VEGF-0089 that are in close proximity.
- FIG. 4 Epitope amino acids bound by VEGF-0089 Fab fragment in a dimer of VEGF-A121 (SEQ ID NO: 36) as determined by X ray crystallography according to Example 3. Amino acid positions comprised in each one of the VEGF-A121 molecules in contact with VEGF-0089 Fab fragment within a distance of 5 ⁇ are highlighted in black.
- FIG. 5 Overlay of the crystal structures of a human VEGF-A121-dimer in complex with VEGF-R1 domain 2 and a human VEGF-A121-dimer in complex with VEGF-0089 Fab as measured in Example 3.
- FIG. 6 Overlay of the crystal structures of a human VEGF-A121-dimer in complex with VEGF-R2 domains 2 and 3 and a human VEGF-A121-dimer in complex with VEGF-0089 Fab as measured in Example 3.
- FIG. 7 Inhibition of VEGF binding to VEGF-R1 in presence of anti-VEGF antibodies as described in Example 4 (0.34 nM VEGF).
- FIG. 8 Inhibition of VEGF binding to VEGF-R1 in presence of anti-VEGF antibodies as described in Example 4 (0.7 nM VEGF).
- FIG. 9 Inhibition of VEGF binding to VEGF-R2 in presence of anti-VEGF antibodies as described in Example 4 (0.34 nM VEGF).
- FIG. 10 Inhibition of VEGF binding to VEGF-R2 in presence of anti-VEGF antibodies as described in Example 4 (0.7 nM VEGF).
- antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
- Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains (VH and VL, respectively) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1).
- Fab fragment thus refers to an antibody fragment comprising a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CH1 domain.
- an “isolated” antibody is one which has been separated from a component of its natural environment.
- an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods.
- electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
- chromatographic e.g., ion exchange or reverse phase HPLC
- antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
- antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
- variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
- the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs), including the complementarity determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)).
- FRs conserved framework regions
- HVRs hypervariable regions
- CDRs complementarity determining regions
- An antigen binding site is formed by several individual amino acid residues from the antibody's heavy and light chain variable domains arranged that are arranged in spatial proximity in the tertiary structure of the Fv region.
- the antigen binding site is defined as a set of the six CDRs comprised in a cognate VH/VL pair.
- CDRs complementarity determining regions
- antibodies comprise six CDRs: three in the VH domain (CDR-H1, CDR-H2, CDR-H3), and three in the VL domain (CDR-L1, CDR-L2, CDR-L3).
- CDR residues and other residues in the variable domain are numbered herein according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
- acceptor human framework for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
- VL light chain variable domain
- VH heavy chain variable domain
- Framework refers to variable domain amino acid residues other than CDR residues.
- the framework of a variable domain generally consists of four framework domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR amino acid sequences generally appear in the following sequence in the (a) VH domain: FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; and (b) in the VL domain: FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4.
- VEGF Vascular endothelial growth factor
- the term encompasses “full-length”, unprocessed VEGF as well as any form of VEGF that results from processing in the cell.
- the term also encompasses naturally occurring variants of VEGF, e.g., splice variants or allelic variants.
- the amino acid sequence of an exemplary human VEGF is shown in SEQ ID NO:33.
- VEGF-dimer refers to a homodimer of two identical VEGF-molecules.
- a complex formed by two identical antibody molecules that are bound to a VEGF-dimer is herein referred to as “VEGF-dimer-antibody-complex”.
- a “first and a second antigen binding site” comprised in a VEGF-dimer-antibody-complex refers to the antigen binding site that is comprised in the VH/VL pair of each one of the two antibodies comprised in the VEGF-dimer-antibody-complex.
- the antigen binding site of one of the two anti-VEGF antibodies in the VEGF-dimer-antibody-complex is the “first antigen binding site”
- the antigen binding site of other one of the two anti-VEGF antibodies is automatically the “second antigen binding site”.
- VEGF vascular endothelial growth factor receptors
- VEGFRs tyrosine kinase receptors
- VEGF-R1 and VEGF-R2 are closely related receptor tyrosine kinases (RTK).
- VEGF-A binds to VEGFR-1 (Flt-1), interacting with domain 2 of VEGF-R1, and VEGFR-2 (KDR/Flk-1), interacting with domains 2 and 3 of VEGF-R2 (see FIG. 1 ).
- VEGF-R1-binding region and “VEGF-R2-binding region” of a VEGF molecule or a VEGF-dimer as used herein refers to those amino acids on the VEGF that interact with domain 2 of VEGF-R1 or domains 2 or 3 of VEGF-R2, respectively.
- anti-VEGF antibody and “an antibody that binds to VEGF” refer to an antibody that is capable of binding VEGF with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting VEGF.
- the extent of binding of an anti-VEGF antibody to an unrelated, non-VEGF protein is less than about 10% of the binding of the antibody to VEGF as measured, e.g., by surface plasmon resonance (SPR).
- SPR surface plasmon resonance
- an antibody that binds to VEGF has a dissociation constant (K D ) of ⁇ 1 nM, or ⁇ 0.15 nM.
- K D dissociation constant
- An antibody is said to “specifically bind” to VEGF when the antibody has a K D of 1 ⁇ M or less.
- VEGFR-blocking selectivity is used herein as an abbreviative term when referred to the property of anti-VEGF antibodies that preferentially inhibit binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1, when bound to a VEGF-dimer.
- Anti-VEGF antibodies that are capable of fully blocking VEGF-binding to VEGF-R2, but not fully block VEGF-binding to VEGF-R1, are condifered to selectively block VEGF-signalling through VEGF-R2 but not through VEGF-R1, i.e. exhibit “VEGFR-blocking selectivity”.
- the “tertiary structure” of a protein is the three dimensional shape of the protein.
- the tertiary structure exhibits a single polypeptide chain “backbone” with one or more protein secondary structures, the protein domains.
- Amino acid side chains may interact and bond in a number of ways. The interactions and bonds of side chains within a particular protein determine its tertiary structure.
- the “tertiary structure of a VEGF-dimer-antibody-complex” as used herein means the threedimensional shape of said complex.
- Amino acid residues located “in close proximity” within a tertiary structure of a VEGF-dimer-antibody-complexes are amino acid residues derived from both anti-VEGF antibodies that are spatially arranged in the threedimensional shape of said complex in a way that their distance is up to 5 ⁇ . This does not include amino acids adjacent to each other in the amino acid sequence of the individual domain, i.e. VH or VL, of the respective anti-VEGF antibody.
- Binding affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described herein.
- epitope denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an anti-VEGF antibody binds.
- Epitopes can be formed both from contiguous amino acid stretches (linear epitope) or comprise non-contiguous amino acids (conformational epitope), e.g. coming in spatial proximity due to the folding of the antigen, i.e. by the tertiary folding of a proteinaceous antigen.
- Linear epitopes are typically still bound by an anti-VEGF antibody after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents.
- An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
- Screening for antibodies binding to a particular epitope can be done using methods routine in the art such as, e.g., without limitation, alanine scanning, peptide blots (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).
- SAP Antigen Structure-based Antibody Profiling
- MAP Modification-Assisted Profiling
- the antibodies in each bin bind to the same epitope which may be a unique epitope either distinctly different from or partially overlapping with epitope represented by another bin.
- competitive binding can be used to easily determine whether an antibody binds to the same epitope of VEGF as, or competes for binding with, a reference anti-VEGF antibody.
- an “antibody that binds to the same epitope” as a reference anti-VEGF antibody refers to an antibody that blocks binding of the reference anti-VEGF antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
- the reference antibody is allowed to bind to VEGF under saturating conditions.
- the ability of an anti-VEGF antibody in question to bind to VEGF is assessed. If the anti-VEGF antibody is able to bind to VEGF after saturation binding of the reference anti-VEGF antibody, it can be concluded that the anti-VEGF antibody in question binds to a different epitope than the reference anti-VEGF antibody. But, if the anti-VEGF antibody in question is not able to bind to VEGF after saturation binding of the reference anti-VEGF antibody, then the anti-VEGF antibody in question may bind to the same epitope as the epitope bound by the reference anti-VEGF antibody.
- two antibodies are deemed to bind to the same or an overlapping epitope if a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50%, at least 75%, at least 90% or even 99% or more as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).
- two antibodies are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other.
- Two antibodies are deemed to have “overlapping epitopes” if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
- nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
- An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
- isolated nucleic acid encoding an antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
- vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
- the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
- Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
- host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
- Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
- Amino acids may be grouped according to common side-chain properties: (1) hydrophobic side chains: Norleucine, Met (M), Ala (A), Val (V), Leu (L), Ile (I); uncharged hydrophilic side chains (also referred to in the art as “neutral” hydrophilic side chains): Cys (C), Ser (S), Thr (T), Asn (N), Gln (Q); negatively charged side chains (also referred to in the art as “acidic” side chains): Asp (D), Glu (E); positively charged side chains (also referred to in the art as “basic” side chains): His (H), Lys (K), Arg (R); aromatic side chains: Trp (W), Tyr (Y), Phe (F); and side chains that comprise residues that influence chain orientation: Gly (G), Pro (P).
- hydrophobic side chains Norleucine, Met (M), Ala (A), Val (V), Leu (L), Ile (I); uncharged hydrophilic side chains (also
- amino acids “having a larger side chain volume” refer to amino acids that have a larger side chain volume than the original amino acid located at the position to be modified.
- amino acids having a larger side chain volume are aromatic amino acids, including tryptophane, tyrosine and phenylalanine.
- the present invention relates to a method of improving VEGFR-blocking selectivity of an antibody that binds to VEGF comprising an antigen binding site formed by cognate pair of a VH and a VL domain, wherein the antibody binds to an epitope of VEGF that overlaps with the VEGF-R1-binding region and the VEGF-R2-binding region in the VEGF molecule.
- the method of the invention comprises (a) providing an analysis of the tertiary structure of a complex of a VEGF-dimer bound by a first and a second antigen binding site of said antibody that binds to VEGF (VEGF-dimer-antibody-complex), (b) identifying at least one amino acid residue located in the VH domain or VL domain of said antibody, wherein said amino acid residue within the first antigen binding site and said amino acid residue within the second antigen binding site are spatially arranged in close proximity in the VEGF-dimer-antigen-complex; and (c) substituting said at least one amino acid residue identified in step (b) by an amino acid having a larger side chain volume.
- an antibody is provided that preferentially inhibits binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1 (VEGFR-blocking selectivity).
- the method of the invention is for improving anti-VEGF antibodies that bind to an epitope of VEGF that overlaps with the VEGF-R1-binding region and the VEGF-R2-binding region in the VEGF molecule.
- said epitope comprises amino acids interacting with domain 2 of VEGF-R1, when VEGF is bound to VEGF-R1.
- said epitope comprises amino acids interacting with domains 2 and 3 of VEGF-R2, when VEGF is bound to VEGF-R1.
- said anti-VEGF antibody binds to an epitope that overlaps with the epitope bound by an antibody characterized by a VH of SEQ ID NO:01 and a VL of SEQ ID NO:02 (antibody VEGF-0089 as described herein). In one embodiment said anti-VEGF antibody binds to the same epitope than antibody VEGF-0089 as described herein, as measured by x-ray crystallography. In one embodiment said anti-VEGF antibody binds to the same epitope than antibody VEGF-0089 as described herein, as measured by x-ray crystallography as described in Example 3.
- said epitope is a conformational epitope within a dimer of VEGF-A121, wherein VEGF-A121 comprises an amino acid sequence of SEQ ID NO: 36, wherein the epitope comprises in one of the individual VEGF-A121 molecules within the VEGF dimer amino acids F17, M18, D19, Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, L66, N100, K101, C102, E103, C104, R105 and P106; and in the other one of the individual VEGF-A121 molecules within the VEGF dimer amino acids E30, K48, M81 and Q87.
- the numbering is according to the position of the amino acid in the amino acid sequence of VEGF-A121 indicated in SEQ ID NO: 36 (see also FIG. 4 ).
- a method of the invention an analysis of the tertiary structure of a VEGF-dimer-antibody-complex is provided.
- the tertiary structure may be provided by methods known in the art.
- the tertiary structure is provided by x-ray crystallography, e.g. as described in Example 3 herein.
- amino acid residues in the VH domain and/or VL domain of said antibody are identified that are in close proximity within the VEGF-dimer-antibody-complex.
- close proximity refers to a distance of 10 ⁇ or less. In one embodiment “close proximity” refers to a distance of 5 ⁇ or less. This at least one amino acid residue is considered suitable for modification.
- such at least one amino acid residue identified in step (b) is located in the heavy chain variable domain of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within a heavy chain CDR of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within H-CDR2 of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within a heavy chain FR of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within H-FR3 of said antibody.
- such amino acid residue is substituted by an amino acid having a larger side chain volume than the amino acid residue comprised in the anti-VEGF antibody to be improved.
- said amino acid having a larger side chain volume is an aromatic amino acid.
- said amino acid having a larger side chain volume is Trp (W), Tyr (Y), or Phe (F).
- said anti-VEGF antibody of step a) binds to human VEGFA. In one embodiment said anti-VEGF antibody of step a) binds to human VEGF of SEQ ID NO:33.
- the anti-VEGF antibody improved by a method of the invention comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:03; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:04; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:05; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:06; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:07; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:08.
- One exemplary antibody comprising this set of CDR amino acid sequences is the antibody referred to herein as “VEGF-0089”.
- said antibody is characterized by a VH comprising SEQ ID NO:01 and a VL comprising SEQ ID NO:02.
- the at least one amino acid residue identified in step (b) is located within H-CDR2 of said antibody.
- said at least one amino acid residue is selected from Kabat position 52a, 52c and 54.
- one, two or three amino acids of Kabat position 52a, 52c and 54 are modified.
- at least one amino acid residue is selected from Kabat position 52a, 52c and 54 is modified by substitutions with Trp (W), Tyr (Y), or Phe (F).
- step c) includes one or more of the following substitutions N52aS, G52cP and I54F.
- the at least one amino acid residue identified in step (b) is located within H-FR3 of said antibody.
- said at least one amino acid residue is Kabat position 74.
- at least the amino acid residue at Kabat position 74 is modified by substitutions with Trp (W), Tyr (Y), or Phe (F).
- step c) includes a substitution S74W.
- the present invention also relates to an anti-VEGF antibody provided by a method of the invention.
- the present invention further relates to an anti-VEGF antibody, wherein binding of the antibody to VEGF significantly inhibits VEGF-binding to VEGF receptor VEGF-R2 without significantly inhibiting VEGF-binding to VEGF receptor VEGF-R1, provided by a method of the invention.
- the antibody provided by a method of the invention is an isolated antibody.
- anti-VEGF antibodies may be produced using recombinant methods known in the art, e.g., as described in U.S. Pat. No. 4,816,567, e.g. recombinant expression in eukaryotic cell such as HEK293 cells as described in Example 1.
- eukaryotic cell such as HEK293 cells as described in Example 1.
- an antibody provided herein is an antibody fragment.
- the antibody fragment is a Fab, Fab′, Fab′-SH, or F(ab′)2 fragment, in particular a Fab fragment.
- an antibody provided herein is a full length antibody.
- the antibody is an IgG1 antibody.
- the present invention further relates to a nucleic acid encoding for an anti-VEGF antibody provided by a method of the invention.
- the present invention further relates to a host cell comprising the nucleic acid of the invention.
- FIG. 1 An overlay of the crystal structures of a human VEGF-dimer in complex with VEGF-R1 domain 2 and VEGF-R3 domains 2 and 3 is depicted in FIG. 1 .
- This superimposition illustrates that both VEGF receptors bind to a highly similar region on the VEGF dimer and that it therefore appears highly challenging to generate antibodies that bind to VEGF that do not inhibit VEGF-binding to both receptors, VEGF-R1 and VEGF-R2, in the same fashion.
- VEGF-R1 and VEGF-R2 In line with this, among the plurality of anti-VEGF antibodies known in the art, only few antibodies were reported to selectively block VEGF-binding to VEGF-R2 rather than VEGF-binding to VEGF-R1.
- Antibody VEGF-0089 as described herein was derived from Roche proprietary transgenic rabbits, expressing a humanized antibody repertoire, upon immunization with a VEGF-derived antigen.
- Transgenic rabbits comprising a human immunoglobulin locus are reported in WO 2000/46251, WO 2002/12437, WO 2005/007696, WO 2006/047367, US 2007/0033661, and WO 2008/027986.
- the animals were housed according to the Appendix A “Guidelines for accommodation and care of animals” in an AAALAC-accredited animal facility. All animal immunization protocols and experiments were approved by the Government of Upper Bavaria (permit number 55.2-1-54-2532-90-14) and performed according to the German Animal Welfare Act and the Directive 2010/63 of the European Parliament and Council.
- rabbits 12-16 week old, were immunized with recombinant human VEGF-121 protein coupled to keyhole limpet hemocyanin (KLH) (produced in house). All animals were immunized with 400 ⁇ g protein, emulsified with complete Freund's adjuvant (CFA), at day 0 by intradermal application, followed by 200 ug protein emulsion at weeks 1, 2, 6, 11 and 14, by alternating intramuscular and subcutaneous injections. Blood was taken at days 4, 5 and 6 post immunizations, starting from the 4th immunization onwards.
- CFA complete Freund's adjuvant
- Serum was prepared for immunogen-specific rabbit-, and human-specific immunoglobulin titer determination by ELISA, and peripheral mononuclear cells were isolated, which were used as a source of antigen-specific B cells in the B cell cloning process.
- PBMC Peripheral Blood Mononuclear Cells
- EDTA containing whole blood was diluted twofold with 1 ⁇ PBS (PAA) before density centrifugation using lympholyte mammal (Cedarlane Laboratories) according to the specifications of the manufacturer.
- the PBMCs were washed twice with 1 ⁇ PBS.
- the PBMCs were seeded on sterile 6-well plates (cell culture grade) to deplete macrophages and monocytes through unspecific adhesion. Each well was filled at maximum with 4 ml medium and up to 6 ⁇ 10e6 PBMCs from the immunized rabbit and were allowed to bind for 1 h at 37° C. in the incubator. The cells in the supernatant (peripheral blood lymphocytes (PBLs)) were used for the antigen panning step.
- PBLs peripheral blood lymphocytes
- the anti-IgG FITC (AbD Serotec) and the anti-huCk PE (Dianova) antibody was used for single cell sorting.
- cells from the depletion and enrichment step were incubated with the anti-IgG FITC and the anti-huCk PE antibody in PBS and incubated for 45 min in the dark at 4° C.
- the PBMCs were washed two fold with ice cold PBS.
- the PBMCs were resuspended in ice cold PBS and immediately subjected to the FACS analyses.
- Propidium iodide in a concentration of 5 ⁇ g/ml (BD Pharmingen) was added prior to the FACS analyses to discriminate between dead and live cells.
- a Becton Dickinson FACSAria equipped with a computer and the FACSDiva software (BD Biosciences) were used for single cell sort.
- the cultivation of the rabbit B cells was performed by a method described by Seeber et al. (S Seeber et al. PLoS One 9 (2), e86184. 2014 Feb. 4). Briefly, single sorted rabbit B cells were incubated in 96-well plates with 200 ⁇ l/well EL-4 B5 medium containing Pansorbin Cells (1:100000) (Calbiochem), 5% rabbit thymocyte supernatant (MicroCoat) and gamma-irradiated murine EL-4 B5 thymoma cells (5 ⁇ 10e5 cells/well) for 7 days at 37° C. in the incubator. The supernatants of the B-cell cultivation were removed for screening and the remaining cells were harvested immediately and were frozen at ⁇ 80° C. in 100 ⁇ l RLT buffer (Qiagen).
- RNA encoding the V domains of the antibodies was isolated.
- PCR-products coding for VH or VL were cloned as cDNA into expression vectors and transiently transformed into HEK-293 cells.
- VEGF-0089 antibody comprising a VH domain of SEQ ID NO:01 and a VL domain of SEQ ID NO:02 was selected.
- This antibody is herein also referred to as antibody “VEGF-0089”.
- the antibody VEGF-0089 was generated as a Fab fragment (herein referred to as “VEGF-0089 Fab fragment” or simply “VEGF-0089 Fab”) having the human VH and VL domains and rabbit derived constant domains of the light chain (CLkappa) and heavy chain (CH1).
- the amino acid sequence of the heavy chain of VEGF-0089 Fab fragment is SEQ ID NO:10.
- the amino acid sequence of the light chain of VEGF-0089 Fab fragment is SEQ ID NO:11.
- VEGF-binding of antibody VEGF-0089 Fab fragment was assessed by surface plasmon resonance (SPR) as described below.
- An anti-His capturing antibody (GE Healthcare 28995056) was immobilized to a Series S Sensor Chip C1 (GE Healthcare 29104990) using standard amine coupling chemistry resulting in a surface densitiy of 500-1000 resonance units (RU).
- As running and dilution buffer HBS-P+(10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) was used, the measurement temperature was set to 25° C. and 37° C., respectively.
- hVEGF-A121 was captured to the surface with resulting capture levels ranging from 5 to 35 RU.
- Dilution series of anti-VEGF antibodies (0.37-30 nM) were injected for 120 s, dissociation was monitored for at least 600 s at a flow rate of 30 ⁇ l/min. The surface was regenerated by injecting 10 mM Glycine pH 1.5 for 60 s. Bulk refractive index differences were corrected by subtracting blank injections and by subtracting the response obtained from the control flow cell without captured hVEGF-A121. Rate constants were calculated using the Langmuir 1:1 binding model within the Biacore Evaluation software.
- the KD of the VEGF-0089 Fab fragment was determined to be 134 pM (at a temperature of 25° C.).
- 384 well streptavidin plates (Nunc/Microcoat #11974998001) were coated with 0.25 ⁇ g/mlbiotinylated VEGF-R1 or 0.5 ⁇ g/ml biotinylated VEGF-R2 (inhouse production, each 25 ⁇ l/well in DPBS (1 ⁇ ) (PAN, #P04-36500)). Plates were incubated for 1 h at room temperature. In parallel, VEGF-121-His (inhouse production) at a concentration of 0.7 nM was incubated with antibodies in different dilutions (12 ⁇ 1:2 dilution steps, starting with a concentration of 500 nM).
- This pre-incubation step was carried out in 384 well PP plates (Weidmann medical technology, #23490-101) in 1 ⁇ OSEP buffer (bidest water, 10 ⁇ , Roche, #11 666 789 001+0.5% Bovine Serum Albumin Fraction V, fatty acid free, Roche, #10 735 086 001+0.05% Tween 20). Plates were incubated for 1 h at room temperature.
- Lucentis® (ranibizumab, heavy chain amino acid sequence of SEQ ID NO:34, light chain amino acid sequence of SEQ ID NO:35) was assessed under the same conditions. The results are shown in FIG. 2 .
- VEGF-0089 Fab fragment is capable of fully blocking VEGF-binding to VEGF-R2.
- VEGF-0089 Fab fragment did not fully block VEGF-binding to VEGF-R1. Consequently, VEGF-0089 Fab fragment is considered to selectively block VEGF-signalling through VEGF-R2 but not through VEGF-R1.
- prior art antibody Lucentis® is capable of fully blocking VEGF-binding to both receptors, VEGF-R2 and VEGF-R1.
- X-ray crystallography of VEGF-0089 Fab fragment in complex with VEGF-A121 was performed as follows:
- VEGF-0089 Fab fragment and human VEGF-A121 were mixed in a 1.1:1 molar ratio. After incubation for 16 hours overnight at 4° C. the complex was purified via gelfiltration chromatography on a Superdex200 (16/600) column in 20 mM IViES, 150 mM NaCl, pH6.5. Fractions containing the dimeric complex were pooled and concentrated to 1.44 mg/ml.
- Crystallization of Dimeric VEGF-A121-VEGF-0089 Fab Complex Initial crystallization trials were performed in sitting drop vapor diffusion setups at 21° C. at a protein concentration of 11.5 mg/ml. Crystals appeared within 1 day out of 0.1 M Tris pH 8.5, 0.2 M LiSO 4 , 1.26 M (NH 4 ) 2 SO 4 . Plate shaped crystals grew in a week to a final size of 150 ⁇ 100 ⁇ 30 ⁇ m. The crystals were directly harvested from the screening plate without any further optimization steps.
- the structure was determined by molecular replacement with PHASER (McCoy, A. J, Grosse-Kunstleve, R. W., Adams, P. D., Storoni, L. C., and Read, R. J. J. Appl. Cryst. 40, 658-674 (2007)) using the coordinates of a related in house structure of a Fab fragment and VEGF as search models.
- Programs from the CCP4 suite Coldlaborative Computational Project, Number 4 Acta Cryst.
- R merge ⁇
- R work ⁇
- FIG. 3 A schematic illustration of the crystal structure of two VEGF-0089 Fab fragments in complex with a human VEGF-A121 dimer is shown in FIG. 3 .
- Amino acid residues in the VEGF-A121 dimer in contact within a distance of 5 ⁇ with antibody VEGF-0089 Fab fragment form the conformational epitope bound by VEGF-0089 Fab on the VEGF-A121 dimer.
- the amino acid sequence of VEGF-A121 is SEQ ID NO: 36.
- An illustration of the amino acids comprised in the epitope on both VEGF-A121 molecules in the VEGF dimer is highlighted in FIG. 4 .
- Antibody VEGF-0089 Fab binds to the following epitope on the VEGF-A121 dimer:
- FIG. 1 An overlay of the crystal structures of a human VEGF-dimer in complex with VEGF-R1 domain 2 and VEGF-R3 domains 2 and 3 is depicted in FIG. 1 . This superimposition illustrates that both VEGF receptors bind to a highly similar region on the VEGF dimer.
- FIG. 5 An overlay of the crystal structures of a human VEGF-dimer in complex with VEGF-R1 domain 2 and in complex with VEGF-0089 Fab fragment is depicted in FIG. 5 .
- This superimposition illustrates that the light chain of the VEGF-0089 antibody of the invention superimposes with domain 2 of VEGF-R1.
- An overlay of the crystal structures of a human VEGF-dimer in complex with VEGF-R2 domains 2 and 3, and in complex with VEGF-0089 Fab fragment is depicted in FIG. 6 .
- This superimposition illustrates that the light chain of the VEGF-0089 antibody of the invention superimposes with domain 2 of VEGF-R2.
- the x-ray data show that the epitope bound by the VEGF-0089 antibody overlaps with the region of VEGF that binds to VEGF-R1 and VEGF-R2. From the structural information provided the expectation would be that binding of the VEGF-0089 antibody to VEGF would result in a similar inhibition of both VEGF-R1 and VEGF-R2 binding to VEGF. This is, surprisingly, not the case as demonstrated above in Example 2 and FIG. 2 . Rather, antibody VEGF-0089 preferentially inhibits binding of VEGF to VEGF-R2 rather than the binding of VEGF to VEGF-R1.
- VEGF-R1 is known to be bound by VEGF with strong affinity
- VEGF-R2 is known to be bound by VEGF with weak affinity.
- VEGF exists in dimeric form, it is believed that the number of antibody molecules bound to the VEGF dimer influence binding to VEGF-R1. It is believed that in case only one antibody molecule is bound to the VEGF dimer, the VEGF dimer is still capable of binding to the strong affinity receptor VEGF-R1 while binding to the low affinity receptor VEGF-R2 is inhibited.
- the VEGF-0089 antibody was modified in order to facilitate that preferentially only one antibody molecule rather than two antibody molecules are capable of simultaneously binding to the VEGF dimer.
- the VEGF-dimer-antibody-complex shown in FIG. 3 it can be seen that two regions within the antibody's VH domain, i.e. H-CDR2 and H-FR3, are in close spatial proximity to each other.
- those regions were modified by amino acid substitutions using large amino acids to replace amino acids with smaller side chain volume in this regions.
- aromatic amino acids were used to replace the amino acid comprised in the original VEGF-0089 amino acid sequence.
- VEGF-0013, VEGF-0014, VEGF-P1AD8675, VEGF-P1AE3520 and VEGF-P1AE3521 comprise amino acid substitutions with aromatic amino acid residues within H-CDR2, H-FR3 or both.
- the listed control antibodies comprise amino acid substitutions that were expected to have no effect or to even have a detrimental effect on VEGFR-blocking selectivity.
- Antibody samples were formulated in 20 mM His/HisCl, 140 mM NaCl, pH 6.0, and were split into three aliquots: one aliquot was re-buffered into PBS, respectively, and two aliquots were kept in the original formulation.
- the PBS aliquot and one His/HisC1 aliquot were incubated for 2 weeks (2 w) at 40° C. (His/NaCl) or 37° C. (PBS) in 1 mg/ml, the PBS sample was incubated further for total 4 weeks (4 w).
- the third control aliquot sample was stored at ⁇ 80° C.
- Proteins were separated depending on their molecular size in solution using a chromatographic gel like TSKgel UP-SW3000.
- a chromatographic gel like TSKgel UP-SW3000.
- protein solution was analyzed regarding their relative content of monomer, high molecular species (e.g. aggregates, dimers, impurities) and low molecular species (e.g. degradation products, impurities).
- 0.2 M Potassium phosphate, 0.25 M KCl, pH 6.2 was used as mobile phase.
- Protein solutions were diluted such as ⁇ 50 ⁇ of protein was injected in a volume of 5 and analyzed with a flow rate of 0.3 ml/min at 25° C. Protein detection was done at 280 nm. Peak definition and peak integration were performed as demonstrated in the typical chromatograms in the product specific information document.
- VEGF-binding activity after stress of improved antibody Fab fragments 2 w/40° C./pH 6.0 2 w/37° C./pH 7.4 4 w/37° C./pH 7.4 VEGF- 101 102 101 0089 Fab VEGF- 103 103 101 0113 Fab VEGF- 99 102 99 0114 Fab VEGF- 101 102 109 P1AD8675 Fab VEGF- 100 102 101 P1AE3520 Fab VEGF- 101 103 99 P1AE3521 Fab VEGF- 102 102 P1AE3519 Fab
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Abstract
Description
- This application is a continuation of International Application No. PCT/EP2018/086468, filed Dec. 21, 2018, the disclosure of which is incorporated herein by reference in its entirety, and which claims priority to European Patent Application No. 17211032.2 filed Dec. 29, 2017.
- The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 12, 2020, is named P35212_US_SequenceListing.txt and is 42.7 kilobytes in size.
- The present invention relates to methods for improving anti-VEGF antibodies in order to provide or improve antibodies that preferentially inhibit binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1; and antibodies provided by said methods.
- Anti-VEGF antibodies that are approved for clinical application, such as Avastin® and Lucentis®, inhibit VEGF-binding to both receptors, VEGF-R1 (FLT-1, fms-like tyrosine kinase) and VEGF-R2 (KDR/FLK-1, fetal liver kinase). VEGF-R1 and VEGF-R2 are closely related receptor tyrosine kinases (RTK). While VEGF-R2 is hypothized to be primarily responsible for VEGF-mediated angiogenesis (Holash, J. et al., Proc Natl Acad Sci USA. 2002 Aug. 20; 99(17):11393-8), VEGF-R1 is known to have other important biological roles unrelated to angiogenesis, e.g. in osteoclast differentiation (Aldridge, S. E. et al., Biochem Biophys Res Commun. 2005 Sep. 30; 335(3):793-8).
- A few anti-VEGF antibodies that preferentially inhibit VEGF binding to VEGF-R2 and but do not significantly inhibit VEGF-binding to VEGF-R1 have been reported, but were not yet clinically successfull (WO200064946 describing an antibody termed “2C3”, WO 2009060198 describing an antibody termed “r84”, and WO 2012089176 describing an antibody termed “L3H6”, EP3006465 describing antibodies termed “HF2-1, HF2-5, HF2-9, and HF2-11”). By blocking VEGF binding to VEGF-R2, but not VEGF-R1, the antibodies are described to have an improved safety profile and do not show common toxicity-related side effects associated with anti-VEGF therapy (Brekken, R. A., et al., Cancer Res. 2000 Sep. 15; 60(18):5117-24; Sullivan, L. A., et al., PLoS One, 2010 Aug. 6; 5(8):e12031).
- There is a need for methods for improving anti-VEGF antibodies, particularly the VEGFR-blocking selectivity, to provide promising clinical antibody candidates.
- The present invention relates to a method of improving VEGFR-blocking selectivity of an antibody that binds to VEGF comprising an antigen binding site formed by cognate pair of a VH and a VL domain, wherein the antibody binds to an epitope of VEGF that overlaps with the VEGF-R1-binding region and the VEGF-R2-binding region in the VEGF molecule. The method of the invention comprises (a) providing an analysis of the tertiary structure of a complex of a VEGF-dimer bound by a first and a second antigen binding site of said antibody that binds to VEGF (VEGF-dimer-antibody-complex), (b) identifying at least one amino acid residue located in the VH domain or VL domain of said antibody, wherein said amino acid residue within the first antigen binding site and said amino acid residue within the second antigen binding site are spatially arranged in close proximity in the VEGF-dimer-antigen-complex; and (c) substituting said at least one amino acid residue identified in step b) by an amino acid having a larger side chain volume.
- With the method of the invention the VEGFR-blocking selectivity of certain anti-VEGF antibodies may be improved by a few modifications in their amino acid sequence, particularly in regions that are not involved in antigen binding. Such antibodies may exhibit an improved safety profile, e.g. by avoiding or reducing side effects caused by blocking VEGF-signalling through VEGF-R1.
- In one embodiment the antibody binds to a conformational epitope on a dimer of VEGF-A121, wherein VEGF-A121 comprises an amino acid sequence of SEQ ID NO: 36, wherein the epitope comprises in one of the individual VEGF-A121 molecules within the VEGF dimer amino acids F17, M18, D19, Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, L66, N100, K101, C102, E103, C104, R105 and P106; and in the other one of the individual VEGF-A121 molecules within the VEGF dimer amino acids E30, K48, M81 and Q87. In one embodiment the epitope is measured by x-ray crystallography.
- In one embodiment the amino acid with a larger side chain volume is an aromatic amino acid.
- In one embodiment the at least one substituted amino acid residue is located in the heavy chain variable domain of said antibody.
- Another aspect of the invention is an antibody that binds to VEGF provided by a method of one of the invention.
- Another aspect of the invention is antibody that binds to VEGF, wherein binding of the antibody to VEGF significantly inhibits VEGF-binding to VEGF receptor VEGF-R2 without significantly inhibiting VEGF-binding to VEGF receptor VEGF-R1, provided by a method of the invention.
- The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
-
FIG. 1 : Crystal structure of VEGF dimer (purple) in complex with VEGF-R1 domain 2 (red) and with VEGF-R2 domain 2 and 3 (blue) -
FIG. 2 : Inhibition of VEGF-binding to VEGF-R1 and VEGF-R2 in presence of antibody Fab fragments (VEGF:VEGF-R2/R1 inhibitition ELISA) as described in Example 2. -
FIG. 3 : Crystal structure of VEGF dimer (purple) in complex with anti-VEGF antibody VEGF-0089 as determined by X ray crystallography according to Example 3. Red circle highlights regions in the VH domain of VEGF-0089 that are in close proximity. -
FIG. 4 : Epitope amino acids bound by VEGF-0089 Fab fragment in a dimer of VEGF-A121 (SEQ ID NO: 36) as determined by X ray crystallography according to Example 3. Amino acid positions comprised in each one of the VEGF-A121 molecules in contact with VEGF-0089 Fab fragment within a distance of 5 Å are highlighted in black. -
FIG. 5 : Overlay of the crystal structures of a human VEGF-A121-dimer in complex with VEGF-R1 domain 2 and a human VEGF-A121-dimer in complex with VEGF-0089 Fab as measured in Example 3. -
FIG. 6 : Overlay of the crystal structures of a human VEGF-A121-dimer in complex with VEGF-R2 domains -
FIG. 7 : Inhibition of VEGF binding to VEGF-R1 in presence of anti-VEGF antibodies as described in Example 4 (0.34 nM VEGF). -
FIG. 8 : Inhibition of VEGF binding to VEGF-R1 in presence of anti-VEGF antibodies as described in Example 4 (0.7 nM VEGF). -
FIG. 9 : Inhibition of VEGF binding to VEGF-R2 in presence of anti-VEGF antibodies as described in Example 4 (0.34 nM VEGF). -
FIG. 10 : Inhibition of VEGF binding to VEGF-R2 in presence of anti-VEGF antibodies as described in Example 4 (0.7 nM VEGF). - Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular, and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.
- Unless otherwise defined herein the term “comprising of” shall include the term “consisting of”.
- The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
- Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains (VH and VL, respectively) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). The term “Fab fragment” thus refers to an antibody fragment comprising a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CH1 domain.
- An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).
- An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
- The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs), including the complementarity determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)).
- A “paratope” or an “antigen binding site”, as used interchangeably herein, refers to a part of an antibody which recognizes and binds to an antigen. An antigen binding site is formed by several individual amino acid residues from the antibody's heavy and light chain variable domains arranged that are arranged in spatial proximity in the tertiary structure of the Fv region. In one embodiment, the antigen binding site is defined as a set of the six CDRs comprised in a cognate VH/VL pair.
- The term “complementarity determining regions” or “CDRs” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and contain antigen-contacting residues. Generally, antibodies comprise six CDRs: three in the VH domain (CDR-H1, CDR-H2, CDR-H3), and three in the VL domain (CDR-L1, CDR-L2, CDR-L3). Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
- An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
- “Framework” or “FR” as used herein refers to variable domain amino acid residues other than CDR residues. The framework of a variable domain generally consists of four framework domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR amino acid sequences generally appear in the following sequence in the (a) VH domain: FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; and (b) in the VL domain: FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4.
- Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors. The term “VEGF”, as used herein, refers to any native VEGF from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed VEGF as well as any form of VEGF that results from processing in the cell. The term also encompasses naturally occurring variants of VEGF, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human VEGF is shown in SEQ ID NO:33. The term “VEGF-dimer” as used to herein refers to a homodimer of two identical VEGF-molecules. A complex formed by two identical antibody molecules that are bound to a VEGF-dimer is herein referred to as “VEGF-dimer-antibody-complex”.
- A “first and a second antigen binding site” comprised in a VEGF-dimer-antibody-complex refers to the antigen binding site that is comprised in the VH/VL pair of each one of the two antibodies comprised in the VEGF-dimer-antibody-complex. For example, while the antigen binding site of one of the two anti-VEGF antibodies in the VEGF-dimer-antibody-complex is the “first antigen binding site”, the antigen binding site of other one of the two anti-VEGF antibodies is automatically the “second antigen binding site”.
- VEGF stimulates cellular responses by binding to tyrosine kinase receptors (the VEGF-receptors, or “VEGFRs”) on the cell surface, causing them to dimerize and become activated through transphosphorylation, although to different sites, times, and extents. VEGF-R1 and VEGF-R2 are closely related receptor tyrosine kinases (RTK). VEGF-A binds to VEGFR-1 (Flt-1), interacting with
domain 2 of VEGF-R1, and VEGFR-2 (KDR/Flk-1), interacting withdomains FIG. 1 ). - The “VEGF-R1-binding region” and “VEGF-R2-binding region” of a VEGF molecule or a VEGF-dimer as used herein refers to those amino acids on the VEGF that interact with
domain 2 of VEGF-R1 ordomains - The terms “anti-VEGF antibody” and “an antibody that binds to VEGF” refer to an antibody that is capable of binding VEGF with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting VEGF. In one embodiment, the extent of binding of an anti-VEGF antibody to an unrelated, non-VEGF protein is less than about 10% of the binding of the antibody to VEGF as measured, e.g., by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to VEGF has a dissociation constant (KD) of <1 nM, or <0.15 nM. An antibody is said to “specifically bind” to VEGF when the antibody has a KD of 1 μM or less.
- By “VEGFR-blocking selectivity” is used herein as an abbreviative term when referred to the property of anti-VEGF antibodies that preferentially inhibit binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1, when bound to a VEGF-dimer. Anti-VEGF antibodies that are capable of fully blocking VEGF-binding to VEGF-R2, but not fully block VEGF-binding to VEGF-R1, are condifered to selectively block VEGF-signalling through VEGF-R2 but not through VEGF-R1, i.e. exhibit “VEGFR-blocking selectivity”.
- The “tertiary structure” of a proteinis the three dimensional shape of the protein. The tertiary structure exhibits a single polypeptide chain “backbone” with one or more protein secondary structures, the protein domains. Amino acid side chains may interact and bond in a number of ways. The interactions and bonds of side chains within a particular protein determine its tertiary structure. The “tertiary structure of a VEGF-dimer-antibody-complex” as used herein means the threedimensional shape of said complex.
- Amino acid residues located “in close proximity” within a tertiary structure of a VEGF-dimer-antibody-complexes are amino acid residues derived from both anti-VEGF antibodies that are spatially arranged in the threedimensional shape of said complex in a way that their distance is up to 5 Å. This does not include amino acids adjacent to each other in the amino acid sequence of the individual domain, i.e. VH or VL, of the respective anti-VEGF antibody.
- “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and 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. Specific illustrative and exemplary embodiments for measuring binding affinity are described herein.
- The term “epitope” denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an anti-VEGF antibody binds. Epitopes can be formed both from contiguous amino acid stretches (linear epitope) or comprise non-contiguous amino acids (conformational epitope), e.g. coming in spatial proximity due to the folding of the antigen, i.e. by the tertiary folding of a proteinaceous antigen. Linear epitopes are typically still bound by an anti-VEGF antibody after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
- Screening for antibodies binding to a particular epitope (i.e., those binding to the same epitope) can be done using methods routine in the art such as, e.g., without limitation, alanine scanning, peptide blots (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).
- Antigen Structure-based Antibody Profiling (ASAP), also known as Modification-Assisted Profiling (MAP), allows to bin a multitude of monoclonal antibodies specifically binding to VEGF based on the binding profile of each of the antibodies from the multitude to chemically or enzymatically modified antigen surfaces (see, e.g., US 2004/0101920). The antibodies in each bin bind to the same epitope which may be a unique epitope either distinctly different from or partially overlapping with epitope represented by another bin.
- Also competitive binding can be used to easily determine whether an antibody binds to the same epitope of VEGF as, or competes for binding with, a reference anti-VEGF antibody. For example, an “antibody that binds to the same epitope” as a reference anti-VEGF antibody refers to an antibody that blocks binding of the reference anti-VEGF antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. Also for example, to determine if an antibody binds to the same epitope as a reference anti-VEGF antibody, the reference antibody is allowed to bind to VEGF under saturating conditions. After removal of the excess of the reference anti-VEGF antibody, the ability of an anti-VEGF antibody in question to bind to VEGF is assessed. If the anti-VEGF antibody is able to bind to VEGF after saturation binding of the reference anti-VEGF antibody, it can be concluded that the anti-VEGF antibody in question binds to a different epitope than the reference anti-VEGF antibody. But, if the anti-VEGF antibody in question is not able to bind to VEGF after saturation binding of the reference anti-VEGF antibody, then the anti-VEGF antibody in question may bind to the same epitope as the epitope bound by the reference anti-VEGF antibody. To confirm whether the antibody in question binds to the same epitope or is just hampered from binding by steric reasons routine experimentation can be used (e.g., peptide mutation and binding analyses using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art). This assay should be carried out in two set-ups, i.e. with both of the antibodies being the saturating antibody. If, in both set-ups, only the first (saturating) antibody is capable of binding to VEGF, then it can be concluded that the anti-VEGF antibody in question and the reference anti-VEGF antibody compete for binding to VEGF.
- In some embodiments two antibodies are deemed to bind to the same or an overlapping epitope if a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50%, at least 75%, at least 90% or even 99% or more as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).
- In some embodiments two antibodies are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies are deemed to have “overlapping epitopes” if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
- An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
- “Isolated nucleic acid encoding” an antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
- The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
- The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
- Amino acids may be grouped according to common side-chain properties: (1) hydrophobic side chains: Norleucine, Met (M), Ala (A), Val (V), Leu (L), Ile (I); uncharged hydrophilic side chains (also referred to in the art as “neutral” hydrophilic side chains): Cys (C), Ser (S), Thr (T), Asn (N), Gln (Q); negatively charged side chains (also referred to in the art as “acidic” side chains): Asp (D), Glu (E); positively charged side chains (also referred to in the art as “basic” side chains): His (H), Lys (K), Arg (R); aromatic side chains: Trp (W), Tyr (Y), Phe (F); and side chains that comprise residues that influence chain orientation: Gly (G), Pro (P).
- When referring to amino acid modifications, amino acids “having a larger side chain volume” refer to amino acids that have a larger side chain volume than the original amino acid located at the position to be modified. In certain embodiments, amino acids having a larger side chain volume are aromatic amino acids, including tryptophane, tyrosine and phenylalanine.
- The present invention relates to a method of improving VEGFR-blocking selectivity of an antibody that binds to VEGF comprising an antigen binding site formed by cognate pair of a VH and a VL domain, wherein the antibody binds to an epitope of VEGF that overlaps with the VEGF-R1-binding region and the VEGF-R2-binding region in the VEGF molecule. The method of the invention comprises (a) providing an analysis of the tertiary structure of a complex of a VEGF-dimer bound by a first and a second antigen binding site of said antibody that binds to VEGF (VEGF-dimer-antibody-complex), (b) identifying at least one amino acid residue located in the VH domain or VL domain of said antibody, wherein said amino acid residue within the first antigen binding site and said amino acid residue within the second antigen binding site are spatially arranged in close proximity in the VEGF-dimer-antigen-complex; and (c) substituting said at least one amino acid residue identified in step (b) by an amino acid having a larger side chain volume. With a method of the invention an antibody is provided that preferentially inhibits binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1 (VEGFR-blocking selectivity).
- The method of the invention is for improving anti-VEGF antibodies that bind to an epitope of VEGF that overlaps with the VEGF-R1-binding region and the VEGF-R2-binding region in the VEGF molecule. In one embodiment said epitope comprises amino acids interacting with
domain 2 of VEGF-R1, when VEGF is bound to VEGF-R1. In one embodiment said epitope comprises amino acids interacting withdomains FIG. 4 ). - Within a method of the invention an analysis of the tertiary structure of a VEGF-dimer-antibody-complex is provided. The tertiary structure may be provided by methods known in the art. In one embodiment the tertiary structure is provided by x-ray crystallography, e.g. as described in Example 3 herein.
- In a method of the invention amino acid residues in the VH domain and/or VL domain of said antibody are identified that are in close proximity within the VEGF-dimer-antibody-complex. In one embodiment “close proximity” refers to a distance of 10 Å or less. In one embodiment “close proximity” refers to a distance of 5 Å or less. This at least one amino acid residue is considered suitable for modification.
- In one embodiment such at least one amino acid residue identified in step (b) is located in the heavy chain variable domain of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within a heavy chain CDR of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within H-CDR2 of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within a heavy chain FR of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within H-FR3 of said antibody.
- In the method of the invention such amino acid residue is substituted by an amino acid having a larger side chain volume than the amino acid residue comprised in the anti-VEGF antibody to be improved. In one embodiment said amino acid having a larger side chain volume is an aromatic amino acid. In one embodiment said amino acid having a larger side chain volume is Trp (W), Tyr (Y), or Phe (F).
- In one embodiment said anti-VEGF antibody of step a) binds to human VEGFA. In one embodiment said anti-VEGF antibody of step a) binds to human VEGF of SEQ ID NO:33.
- In one aspect the anti-VEGF antibody improved by a method of the invention comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:03; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:04; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:05; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:06; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:07; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:08. One exemplary antibody comprising this set of CDR amino acid sequences is the antibody referred to herein as “VEGF-0089”. In one embodiment said antibody is characterized by a VH comprising SEQ ID NO:01 and a VL comprising SEQ ID NO:02.
- In one embodiment of this aspect, the at least one amino acid residue identified in step (b) is located within H-CDR2 of said antibody. In one embodiment said at least one amino acid residue is selected from
Kabat position 52a, 52c and 54. In one embodiment one, two or three amino acids ofKabat position 52a, 52c and 54 are modified. In one embodiment at least one amino acid residue is selected fromKabat position 52a, 52c and 54 is modified by substitutions with Trp (W), Tyr (Y), or Phe (F). In one embodiment step c) includes one or more of the following substitutions N52aS, G52cP and I54F. - In one embodiment of this aspect, the at least one amino acid residue identified in step (b) is located within H-FR3 of said antibody. In one embodiment said at least one amino acid residue is
Kabat position 74. In one embodiment at least the amino acid residue atKabat position 74 is modified by substitutions with Trp (W), Tyr (Y), or Phe (F). In one embodiment step c) includes a substitution S74W. - The present invention also relates to an anti-VEGF antibody provided by a method of the invention. The present invention further relates to an anti-VEGF antibody, wherein binding of the antibody to VEGF significantly inhibits VEGF-binding to VEGF receptor VEGF-R2 without significantly inhibiting VEGF-binding to VEGF receptor VEGF-R1, provided by a method of the invention. In one embodiment the antibody provided by a method of the invention is an isolated antibody.
- Such anti-VEGF antibodies may be produced using recombinant methods known in the art, e.g., as described in U.S. Pat. No. 4,816,567, e.g. recombinant expression in eukaryotic cell such as HEK293 cells as described in Example 1. For these methods one or more isolated nucleic acid(s) encoding an antibody are provided.
- In certain embodiments, an antibody provided herein is an antibody fragment. In one embodiment, the antibody fragment is a Fab, Fab′, Fab′-SH, or F(ab′)2 fragment, in particular a Fab fragment.
- In certain embodiments, an antibody provided herein is a full length antibody.
- In one embodiment the antibody is an IgG1 antibody.
- The present invention further relates to a nucleic acid encoding for an anti-VEGF antibody provided by a method of the invention. The present invention further relates to a host cell comprising the nucleic acid of the invention.
-
DESCRIPTION OF THE AMINO ACID SEQUENCES SEQ ID NO: 1 VH domain of antibody VEGF-0089 EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGNGGGIYTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 2 VL domain of antibodies VEGF-0089, VEGF-0113, VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520, VEGF-P1AE3521, VEGF-0112, VEGF-P1AE8674, VEGF-P1AE3519 DIQMTQSPASLSASVGDRVTITCRASQSIYSSLNWYQQKPGK APKLLIYASTLQSGVPSRFSGSASGTDFTLTISSLQPEDVAT YYCQQYQNFPRTFGQGTKLEIK SEQ ID NO: 3 H-CDR1 of antibodies VEGF-0089, VEGF-0113, VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520, VEGF-P1AE3521, VEGF-0112, VEGF-P1AE8674, VEGF-P1AE3519 NYAMT SEQ ID NO: 4 H-CDR2 of antibodies VEGF-0089, VEGF-P1AD8675, VEGF-P1AD8674 SIGNG G GIYTYYADSVKG SEQ ID NO: 5 H-CDR3 of antibodies VEGF-0089, VEGF-0113, VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520, VEGF-P1AE3521, VEGF-0112, VEGF-P1AE8674, VEGF-P1AE3519 GDNLFDS SEQ ID NO: 6 L-CDR1 of antibodies VEGF-0089, VEGF-0113, VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520, VEGF-P1AE3521,VEGF-0112, VEGF-P1AE8674, VEGF-P1AE3519 RASQSIYSSLN SEQ ID NO: 7 L-CDR2 of antibodies VEGF-0089, VEGF-0113, VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520, VEGF-P1AE3521, VEGF-0112, VEGF-P1AE8674, VEGF-P1AE3519 ASTLQSGVPSR SEQ ID NO: 8 L-CDR3 of antibodies VEGF-0089, VEGF-0113, VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520, VEGF-P1AE3521, VEGF-0112, VEGF-P1AE8674, VEGF-P1AE3519 FPRT SEQ ID NO: 9 H-FR3 of VEGF-0089, VEGF-0113,VEGF-0114, VEGF-0112 FTISRDN S KNTLYLQMNSLRAEDTAVYYCAK SEQ ID NO: 10 heavy chain of VEGF-0089 Fab fragment EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGNGGGIYTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVF PLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVR TFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEA SEQ ID NO: 11 light chain of VEGF-0089, VEGF-0113, VEGF-0114, VEGF-P1AD8675 Fab fragment DIQMTQSPASLSASVGDRVTITCRASQSIYSSLNWYQQKPGK APKLLIYASTLQSGVPSRFSGSASGTDFTLTISSLQPEDVAT YYCQQYQNFPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC SEQ ID NO: 12 VH domain of antibody VEGF-0113 EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGNGPGIYTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 13 H-CDR2 of antibody VEGF-0113, P1AE3520 SIGNGPGIYTYYADSVKG SEQ ID NO: 14 heavy chain of VEGF-0113 Fab fragment EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGNGPGIYTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVF PLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVR TFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEA SEQ ID NO: 15 VH domain of antibody VEGF-0114 EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGSGGFYTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 16 H-CDR2 of antibody VEGF-0114, VEGF-P1AE3521 SIG S GG -F YTYYADSVKG SEQ ID NO: 17 heavy chain of VEGF-0114 Fab fragment EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGSG_GFYTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVF PLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVR TFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEA SEQ ID NO: 18 VH domain of antibody VEGF-P1AD8675 EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGNGGGIYTYYADSVKGRFTISRDNWKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 19 H-FR3 of antibody VEGF-P1AD8675, P1AE3520, VEGF-P1AE3521 FTISRDN W KNTLYLQMNSLRAEDTAVYYCAK SEQ ID NO: 20 heavy chain of VEGF-P1AD8675 Fab fragment EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGNGGGIYTYYADSVKGRFTISRDNWKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSASTKGPSVF PLAPSSKSISGGTAALGCLVKDYFPEPVTVSWNSGALISGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSC SEQ ID NO: 21 VH domain of antibody VEGF-P1AE3520 EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGNGPGIYTYYADSVKGRFTISRDNWKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 22 heavy chain of VEGF-P1AE3520 Fab fragment EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGNGPGIYTYYADSVKGRFTISRDNWKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVF PLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVR TFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEA SEQ ID NO: 23 VH domain of antibody VEGF-P1AE3521 EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGSGGFYTYYADSVKGRFTISRDNWKNTLYLQMN SLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 24 heavy chain of VEGF-P1AE3521 Fab fragment EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGSGGFYTYYADSVKGRFTISRDNWKNTLYLQMN SLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVFP LAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRT FPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKT VAPSTCSEQKLISEEDLGAAEPEA SEQ ID NO: 25 VH domain of antibody VEGF-0112 EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGSGGGIYTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 26 H-CDR2 of antibody VEGF-0112 SIGSGGGIYTYYADSVKG SEQ ID NO: 27 heavy chain of VEGF-0112 Fab fragment EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGSGGGIYTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVF PLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVR TFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEA SEQ ID NO: 28 VH domain of antibody VEGF-P1AD8674 EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGNGGGIYTYYADSVKGRFTISRNNAENTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 29 H-FR3 of antibody VEGF-P1AD8674 FTISRNNAENTLYLQMNSLRAEDTAVYYCAK SEQ ID NO: 30 heavy chain of VEGF-P1AD8674 Fab fragment EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGNGGGIYTYYADSVKGRFTISRNNAENTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSASTKGPSVF PLAPSSKSISGGTAALGCLVKDYFPEPVTVSWNSGALISGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSC SEQ ID NO: 31 VH domain of antibody VEGF-P1AE3519 EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGSGGGIYTYYADSVKGRFTISRDNWKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 32 heavy chain of VEGF-P1AE3519 Fab fragment EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPG KGLEWVSSIGSGGGIYTYYADSVKGRFTISRDNWKNTLYLQM NSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVF PLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVR TFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEA SEQ ID NO: 33 human VEGF MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVK FMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCG GCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHN KCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVG ARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTD SRCKARQLELNERTCRCDKPRR SEQ ID NO: 34 heavy chain of Lucentis (ranibizumab) DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGK APKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC SEQ ID NO: 35 light chain of Lucentis (ranibizumab) EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPG KGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMN SLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSC SEQ ID NO: 36 Human VEGF-A121 APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDE IEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIK PHQGQHIGEMSFLQHNKCECRPKKDRARQEKCDKPRR - The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.
- An overlay of the crystal structures of a human VEGF-dimer in complex with VEGF-
R1 domain 2 and VEGF-R3 domains FIG. 1 . This superimposition illustrates that both VEGF receptors bind to a highly similar region on the VEGF dimer and that it therefore appears highly challenging to generate antibodies that bind to VEGF that do not inhibit VEGF-binding to both receptors, VEGF-R1 and VEGF-R2, in the same fashion. In line with this, among the plurality of anti-VEGF antibodies known in the art, only few antibodies were reported to selectively block VEGF-binding to VEGF-R2 rather than VEGF-binding to VEGF-R1. - Antibody VEGF-0089 as described herein was derived from Roche proprietary transgenic rabbits, expressing a humanized antibody repertoire, upon immunization with a VEGF-derived antigen. Transgenic rabbits comprising a human immunoglobulin locus are reported in WO 2000/46251, WO 2002/12437, WO 2005/007696, WO 2006/047367, US 2007/0033661, and WO 2008/027986. The animals were housed according to the Appendix A “Guidelines for accommodation and care of animals” in an AAALAC-accredited animal facility. All animal immunization protocols and experiments were approved by the Government of Upper Bavaria (permit number 55.2-1-54-2532-90-14) and performed according to the German Animal Welfare Act and the Directive 2010/63 of the European Parliament and Council.
- Briefly, rabbits (n=3), 12-16 week old, were immunized with recombinant human VEGF-121 protein coupled to keyhole limpet hemocyanin (KLH) (produced in house). All animals were immunized with 400 μg protein, emulsified with complete Freund's adjuvant (CFA), at
day 0 by intradermal application, followed by 200 ug protein emulsion atweeks days - B Cell Cloning from Transgenic Rabbits
- Isolation of Rabbit Peripheral Blood Mononuclear Cells (PBMC):
- Blood samples were taken of immunized rabbits. EDTA containing whole blood was diluted twofold with 1×PBS (PAA) before density centrifugation using lympholyte mammal (Cedarlane Laboratories) according to the specifications of the manufacturer. The PBMCs were washed twice with 1×PBS.
- EL-4 B5 Medium:
- RPMI 1640 (Pan Biotech) supplemented with 10% FCS (Pan Biotech), 2 mM Glutamin, 1% penicillin/streptomycin solution (Gibco), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech) and 0.05 mM b-mercaptoethanole (Invitrogen) was used.
- Coating of Plates:
- Sterile cell culture 6-well plates were coated with 2 μg/ml KLH in carbonate buffer (0.1 M sodium bicarbonate, 34 mM Disodiumhydrogencarbonate, pH 9.55) over night at 4° C. Plates were washed in sterile PBS three times before use.
- Depletion of Macrophages/Monocytes or of Human Fc Binders:
- The PBMCs were seeded on sterile 6-well plates (cell culture grade) to deplete macrophages and monocytes through unspecific adhesion. Each well was filled at maximum with 4 ml medium and up to 6×10e6 PBMCs from the immunized rabbit and were allowed to bind for 1 h at 37° C. in the incubator. The cells in the supernatant (peripheral blood lymphocytes (PBLs)) were used for the antigen panning step.
- Immune Fluorescence Staining and Flow Cytometry:
- The anti-IgG FITC (AbD Serotec) and the anti-huCk PE (Dianova) antibody was used for single cell sorting. For surface staining, cells from the depletion and enrichment step were incubated with the anti-IgG FITC and the anti-huCk PE antibody in PBS and incubated for 45 min in the dark at 4° C. After staining the PBMCs were washed two fold with ice cold PBS. Finally the PBMCs were resuspended in ice cold PBS and immediately subjected to the FACS analyses. Propidium iodide in a concentration of 5 μg/ml (BD Pharmingen) was added prior to the FACS analyses to discriminate between dead and live cells. A Becton Dickinson FACSAria equipped with a computer and the FACSDiva software (BD Biosciences) were used for single cell sort.
- B-Cell Cultivation:
- The cultivation of the rabbit B cells was performed by a method described by Seeber et al. (S Seeber et al. PLoS One 9 (2), e86184. 2014 Feb. 4). Briefly, single sorted rabbit B cells were incubated in 96-well plates with 200 μl/well EL-4 B5 medium containing Pansorbin Cells (1:100000) (Calbiochem), 5% rabbit thymocyte supernatant (MicroCoat) and gamma-irradiated murine EL-4 B5 thymoma cells (5×10e5 cells/well) for 7 days at 37° C. in the incubator. The supernatants of the B-cell cultivation were removed for screening and the remaining cells were harvested immediately and were frozen at −80° C. in 100 μl RLT buffer (Qiagen).
- RNA encoding the V domains of the antibodies was isolated. For recombinant expression of the antibody PCR-products coding for VH or VL were cloned as cDNA into expression vectors and transiently transformed into HEK-293 cells.
- From the screening an antibody comprising a VH domain of SEQ ID NO:01 and a VL domain of SEQ ID NO:02 was selected. This antibody is herein also referred to as antibody “VEGF-0089”. For the subsequent analyses the antibody VEGF-0089 was generated as a Fab fragment (herein referred to as “VEGF-0089 Fab fragment” or simply “VEGF-0089 Fab”) having the human VH and VL domains and rabbit derived constant domains of the light chain (CLkappa) and heavy chain (CH1). The amino acid sequence of the heavy chain of VEGF-0089 Fab fragment is SEQ ID NO:10. The amino acid sequence of the light chain of VEGF-0089 Fab fragment is SEQ ID NO:11.
- VEGF-binding of antibody VEGF-0089 Fab fragment was assessed by surface plasmon resonance (SPR) as described below.
- An anti-His capturing antibody (GE Healthcare 28995056) was immobilized to a Series S Sensor Chip C1 (GE Healthcare 29104990) using standard amine coupling chemistry resulting in a surface densitiy of 500-1000 resonance units (RU). As running and dilution buffer, HBS-P+(10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) was used, the measurement temperature was set to 25° C. and 37° C., respectively. hVEGF-A121 was captured to the surface with resulting capture levels ranging from 5 to 35 RU. Dilution series of anti-VEGF antibodies (0.37-30 nM) were injected for 120 s, dissociation was monitored for at least 600 s at a flow rate of 30 μl/min. The surface was regenerated by injecting 10 mM Glycine pH 1.5 for 60 s. Bulk refractive index differences were corrected by subtracting blank injections and by subtracting the response obtained from the control flow cell without captured hVEGF-A121. Rate constants were calculated using the Langmuir 1:1 binding model within the Biacore Evaluation software.
- As a result, the KD of the VEGF-0089 Fab fragment was determined to be 134 pM (at a temperature of 25° C.).
- For further characterization of the antibody, inhibition of VEGF-binding to its receptors VEGF-R1 and VEGF-R2 in presence of VEGF-0089 Fab fragment was assessed as described below:
- 384 well streptavidin plates (Nunc/Microcoat #11974998001) were coated with 0.25 μg/mlbiotinylated VEGF-R1 or 0.5 μg/ml biotinylated VEGF-R2 (inhouse production, each 25 μl/well in DPBS (1×) (PAN, #P04-36500)). Plates were incubated for 1 h at room temperature. In parallel, VEGF-121-His (inhouse production) at a concentration of 0.7 nM was incubated with antibodies in different dilutions (12×1:2 dilution steps, starting with a concentration of 500 nM). This pre-incubation step was carried out in 384 well PP plates (Weidmann medical technology, #23490-101) in 1×OSEP buffer (bidest water, 10×, Roche, #11 666 789 001+0.5% Bovine Serum Albumin Fraction V, fatty acid free, Roche, #10 735 086 001+0.05% Tween 20). Plates were incubated for 1 h at room temperature. After washing VEGF-R1/VEGF-R2 coated
streptavidin plates 3 times with 90 μl/well PBST-buffer (bidest water, 10×PBS Roche #11666789001+0.1% Tween 20), 25 μl of samples from the VEGF-antibody pre-incubation plate were transferred to coated strepavidin plates which were subsequenty incubated for 1 h at room temperature. After washing 3 times with 90 μl/well PBST-buffer, 25 μl/well detection antibody (anti His POD, Bethyl, #A190-114P, 1:12000) in 1×OSEP was added. After incubation for 1 h at room temperature plates were washed 3 times with 90 μl PBST-buffer. 25 μl TMB (Roche, #11 835 033 001) was added to all wells simultaneously. After 10 min incubation at room temperature, signals were detected at 370 nm/492 nm on aTecan Safire 2 Reader. - As a control and representative for a prior art anti-VEGF antibody that is used in the clinic, Lucentis® (ranibizumab, heavy chain amino acid sequence of SEQ ID NO:34, light chain amino acid sequence of SEQ ID NO:35) was assessed under the same conditions. The results are shown in
FIG. 2 . - The results indicate that VEGF-0089 Fab fragment is capable of fully blocking VEGF-binding to VEGF-R2. VEGF-0089 Fab fragment did not fully block VEGF-binding to VEGF-R1. Consequently, VEGF-0089 Fab fragment is considered to selectively block VEGF-signalling through VEGF-R2 but not through VEGF-R1. As illustrated in
FIG. 2 , prior art antibody Lucentis® is capable of fully blocking VEGF-binding to both receptors, VEGF-R2 and VEGF-R1. - X-Ray Crystallography of Antibody VEGF-0089 in Complex with VEGF-Dimer and Epitope Determination
- The crystal structure of VEGF-0089 Fab fragment as described above was analyzed according to standard methods known in the art.
- X-ray crystallography of VEGF-0089 Fab fragment in complex with VEGF-A121 was performed as follows:
- Complex Formation and Purification of the Dimeric Complex VEGF-A121-VEGF-0089 Fab.
- For complex formation the VEGF-0089 Fab fragment and human VEGF-A121 (Peprotech) were mixed in a 1.1:1 molar ratio. After incubation for 16 hours overnight at 4° C. the complex was purified via gelfiltration chromatography on a Superdex200 (16/600) column in 20 mM IViES, 150 mM NaCl, pH6.5. Fractions containing the dimeric complex were pooled and concentrated to 1.44 mg/ml.
- Crystallization of Dimeric VEGF-A121-VEGF-0089 Fab Complex. Initial crystallization trials were performed in sitting drop vapor diffusion setups at 21° C. at a protein concentration of 11.5 mg/ml. Crystals appeared within 1 day out of 0.1 M Tris pH 8.5, 0.2 M LiSO4, 1.26 M (NH4)2SO4. Plate shaped crystals grew in a week to a final size of 150×100×30 μm. The crystals were directly harvested from the screening plate without any further optimization steps.
- Data Collection and Structure Determination.
- For data collection crystals were flash cooled at 100K in precipitant solution with addition of 15% ethylene glycol as cryoprotectant. Diffraction data were collected at a wavelength of 1.0000 Å using a PILATUS 6M detector at the beamline X10SA of the Swiss Light Source (Villigen, Switzerland). Data have been processed with XDS (Kabsch, W. Acta Cryst. D66, 133-144 (2010)) and scaled with SADABS (BRUKER). The crystals belong to the space group C2 with cell axes of a=227.61 Å, b=66.97 Å, c=218.31 Å, β=104.54° and diffract to a resolution of 2.17 Å. The structure was determined by molecular replacement with PHASER (McCoy, A. J, Grosse-Kunstleve, R. W., Adams, P. D., Storoni, L. C., and Read, R. J. J. Appl. Cryst. 40, 658-674 (2007)) using the coordinates of a related in house structure of a Fab fragment and VEGF as search models. Programs from the CCP4 suite (Collaborative Computational Project,
Number 4 Acta Cryst. D50, 760-763 (1994)) and Buster (Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O. S., Vonrhein, C., Womack, T. O. (2011). Buster version 2.9.5 Cambridge, United Kingdom: Global Phasing Ltd) have been used to subsequently refine the data. Manual rebuilding of protein using difference electron density was done with COOT (Emsley, P., Lohkamp, B., Scott, W. G. and Cowtan, K. Acta Cryst D66, 486-501 (2010)). Data collection and refinement statistics for both structures are summarized in the following Table. All graphical presentations were prepared with PYMOL (DeLano Scientific, Palo Alto, Calif., 2002). -
TABLE Data collection and structure refinement statistics Data Collection Wavelength (Å) 1.0 Resolution1 (Å) 49.49-2.17 (2.27-2.17) Space group C2 Unit cell (Å, °) 227.61 66.97 218.31, 90.00 104.54 90.00 Unique reflections 168745 (21164) Multiplicity 3.45 (3.43) Completeness (%) 99.8 (99.6) Mean I/σ(I) 8.36 (0.71) R-meas 0.073 (0.86) CC1/2 0.999 (0.364) Refinement Resolution1 (Å) 49.49-2.17 (2.23-2.17) Reflections used in refinement 168674 (12361) Reflections used for R-free 8487 (617) R-work3 0.185 (0.262) R-free4 0.227 (0.287) Number of atoms 16966 Protein residues 1466 RMS bonds (Å) 0.010 RMS angles (°) 1.20 Ramachandran favored (%) 97.85 Ramachandran outliers (%) 0.15 Rotamer outliers (%) 3.47 Clashscore 2.39 Average B-factor (Å2) 65.89 protein 66.85 solvent 64.05 1Values in parentheses refer to the highest resolution bins. 2Rmerge = Σ|I−<I>|/ΣI where I is intensity. 3Rwork = Σ|Fo−<Fc>|/ΣFo where Fo is the observed and Fc is the calculated structure factor amplitude. 4Rfree was calculated based on 5% of the total data omitted during refinement. - A schematic illustration of the crystal structure of two VEGF-0089 Fab fragments in complex with a human VEGF-A121 dimer is shown in
FIG. 3 . Amino acid residues in the VEGF-A121 dimer in contact within a distance of 5 Å with antibody VEGF-0089 Fab fragment form the conformational epitope bound by VEGF-0089 Fab on the VEGF-A121 dimer. The amino acid sequence of VEGF-A121 is SEQ ID NO: 36. An illustration of the amino acids comprised in the epitope on both VEGF-A121 molecules in the VEGF dimer is highlighted inFIG. 4 . - Antibody VEGF-0089 Fab binds to the following epitope on the VEGF-A121 dimer:
-
- in one of the individual VEGF-A121 molecules within the VEGF dimer amino acids F17, M18, D19, Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, L66, N100, K101, C102, E103, C104, R105 and P106; and
- in the other one of the individual VEGF-A121 molecules within the VEGF dimer amino acids E30, K48, M81 and Q87.
- An overlay of the crystal structures of a human VEGF-dimer in complex with VEGF-
R1 domain 2 and VEGF-R3 domains FIG. 1 . This superimposition illustrates that both VEGF receptors bind to a highly similar region on the VEGF dimer. - An overlay of the crystal structures of a human VEGF-dimer in complex with VEGF-
R1 domain 2 and in complex with VEGF-0089 Fab fragment is depicted inFIG. 5 . This superimposition illustrates that the light chain of the VEGF-0089 antibody of the invention superimposes withdomain 2 of VEGF-R1. An overlay of the crystal structures of a human VEGF-dimer in complex with VEGF-R2 domains FIG. 6 . This superimposition illustrates that the light chain of the VEGF-0089 antibody of the invention superimposes withdomain 2 of VEGF-R2. - Consequently, the x-ray data show that the epitope bound by the VEGF-0089 antibody overlaps with the region of VEGF that binds to VEGF-R1 and VEGF-R2. From the structural information provided the expectation would be that binding of the VEGF-0089 antibody to VEGF would result in a similar inhibition of both VEGF-R1 and VEGF-R2 binding to VEGF. This is, surprisingly, not the case as demonstrated above in Example 2 and
FIG. 2 . Rather, antibody VEGF-0089 preferentially inhibits binding of VEGF to VEGF-R2 rather than the binding of VEGF to VEGF-R1. - Without being bound to this theory, this observation may be resulting from different affinities of VEGF towards its two receptors, VEGF-R1 and VEGF-R2. While VEGF-R1 is known to be bound by VEGF with strong affinity, it is known that VEGF-R2 is known to be bound by VEGF with weak affinity. As VEGF exists in dimeric form, it is believed that the number of antibody molecules bound to the VEGF dimer influence binding to VEGF-R1. It is believed that in case only one antibody molecule is bound to the VEGF dimer, the VEGF dimer is still capable of binding to the strong affinity receptor VEGF-R1 while binding to the low affinity receptor VEGF-R2 is inhibited.
- Based on the theory described in Example 3, the VEGF-0089 antibody was modified in order to facilitate that preferentially only one antibody molecule rather than two antibody molecules are capable of simultaneously binding to the VEGF dimer. From the tertiary structure of the VEGF-dimer-antibody-complex shown in
FIG. 3 it can be seen that two regions within the antibody's VH domain, i.e. H-CDR2 and H-FR3, are in close spatial proximity to each other. To avoid simultaneous binding of two antibody molecules to the VEGF-dimer those regions were modified by amino acid substitutions using large amino acids to replace amino acids with smaller side chain volume in this regions. In the examples aromatic amino acids were used to replace the amino acid comprised in the original VEGF-0089 amino acid sequence. The theory was, that in this case binding of two antibody molecules to a VEGF dimer would be sterically hindered and the observed preferential inhibition of VEGF-binding to VEGF-R2 rather than the VEGF-binding to VEGF-R1 would be even more prominent. - Based on this theory, the following antibody variants of antibody VEGF-0089 were generated. Candidate antibodies VEGF-0013, VEGF-0014, VEGF-P1AD8675, VEGF-P1AE3520 and VEGF-P1AE3521 comprise amino acid substitutions with aromatic amino acid residues within H-CDR2, H-FR3 or both. The listed control antibodies comprise amino acid substitutions that were expected to have no effect or to even have a detrimental effect on VEGFR-blocking selectivity.
-
TABLE 1 Amino acid sequences and amino acid modifications in generated candidate and control antibodies SEQ ID NO: H-CDR2 H-FR3 VII VL VEGF-0089 wt wt 1 2 Candidate antibodies VEGF-0113 G52cP wt 12 2 VEGF-0114 N52aS, G52c(−), wt 15 2 I54F VEGF- P1AD8675 wt S74W 18 2 VEGF- P1AE3520 G52cP S74W 21 2 VEGF-P1AE3521 N52aS, G52c(−), S74W 23 2 I54F Control antibodies VEGF-0112 N52aS wt 25 2 VEGF-P1AD8674 wt D72N, S74A, 28 2 K75E VEGF- P1AE3519 N52aS S74W 31 2
Fab fragments of the antibodies were cloned and expressed as described in Example 1. Amino acid sequences of heavy chains and light chains are shown in Table 2. -
TABLE 2 Amino acid sequences of anti-VEGF antibodies heavy chain light chain VEGF-0089 Fab SEQ ID NO: 10 SEQ ID NO: 11 VEGF-0113 Fab SEQ ID NO: 14 SEQ ID NO: 11 VEGF-0114 Fab SEQ ID NO: 17 SEQ ID NO: 11 VEGF-P1AD8675 Fab SEQ ID NO: 20 SEQ ID NO: 11 VEGF-P1AE3520 Fab SEQ ID NO: 22 SEQ ID NO: 11 VEGF-P1AE3521 Fab SEQ ID NO: 24 SEQ ID NO: 11 VEGF-0112 Fab SEQ ID NO: 27 SEQ ID NO: 11 VEGF-P1AD8674 Fab SEQ ID NO: 30 SEQ ID NO: 11 VEGF-P1AE3519 Fab SEQ ID NO: 32 SEQ ID NO: 11 - Binding of VEGF to VEGF-R1 as well as VEGF-R2 in presence of improved antibody Fab fragments was tested as described in Example 2. All antibody Fab fragments listed in Table 2 were tested. Results are shown in
FIGS. 7 to 10 . - The affinity of the antibodies was determined by SPR using the same methods as described in Example 2. All antibody Fab fragments listed in Table 2 were tested. Results are shown in Table 3.
-
TABLE 3 Affinities of anti-VEGF antibodies KD [pM] at 25° C. KD [pM] at 37° C. VEGF-0089 Fab 143 110 VEGF-0113 Fab 22 29 VEGF-0114 Fab 50 54 VEGF- P1AD8675 Fab 88 65 VEGF- P1AE3520 Fab 31 39 VEGF- P1AE3521 Fab 32 61 VEGF- P1AE3519 Fab 119 94 - Chemical stability of improved antibody Fab fragments was tested as follows:
- Antibody samples were formulated in 20 mM His/HisCl, 140 mM NaCl, pH 6.0, and were split into three aliquots: one aliquot was re-buffered into PBS, respectively, and two aliquots were kept in the original formulation. The PBS aliquot and one His/HisC1 aliquot were incubated for 2 weeks (2 w) at 40° C. (His/NaCl) or 37° C. (PBS) in 1 mg/ml, the PBS sample was incubated further for
total 4 weeks (4 w). The third control aliquot sample was stored at −80° C. After incubation ended, samples were analyzed for relative active concentration (Biacore; active concentration of both stressed aliquots of each binder is normalized to unstressed 4° C. aliquot), aggregation (SEC) and fragmentation (capillary electrophoresis or SDS-PAGE) and compared with the untreated control. - For Size Exclusion UHPLC (=SEC), proteins were separated depending on their molecular size in solution using a chromatographic gel like TSKgel UP-SW3000. With this method, protein solution was analyzed regarding their relative content of monomer, high molecular species (e.g. aggregates, dimers, impurities) and low molecular species (e.g. degradation products, impurities). 0.2 M Potassium phosphate, 0.25 M KCl, pH 6.2 was used as mobile phase. Protein solutions were diluted such as ˜50μ of protein was injected in a volume of 5 and analyzed with a flow rate of 0.3 ml/min at 25° C. Protein detection was done at 280 nm. Peak definition and peak integration were performed as demonstrated in the typical chromatograms in the product specific information document.
- All antibody Fab fragments listed in Table 2 were tested. Results are shown in Tables 4 and 5.
-
TABLE 4 VEGF-binding activity after stress of improved antibody Fab fragments 2 w/40° C./pH 6.0 2 w/37° C./pH 7.4 4 w/37° C./pH 7.4 VEGF- 101 102 101 0089 Fab VEGF- 103 103 101 0113 Fab VEGF- 99 102 99 0114 Fab VEGF- 101 102 109 P1AD8675 Fab VEGF- 100 102 101 P1AE3520 Fab VEGF- 101 103 99 P1AE3521 Fab VEGF- 102 102 102 P1AE3519 Fab -
TABLE 5 Molecular integrity after stress (4 weeks, pH 7.4, 37° C.) of improved antibody Fab fragments Aggregation Main fraction [% aggregates] [%] VEGF-0089 Fab 0.6 99.4 VEGF-0113 Fab 0.6 99.4 VEGF-0114 Fab 2 97.1 VEGF-P1AD8675 Fab 65.3 34.7 VEGF-P1AE3520 Fab 11.9 87.2 VEGF-P1AE3521 Fab 4.8 94.2 VEGF- P1AE3519 Fab 3 96.4
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007054816A2 (en) * | 2005-11-14 | 2007-05-18 | Bioren, Inc. | Antibody ultrahumanization by predicted mature cdr blasting and cohort library generation and screening |
Family Cites Families (97)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
US4676980A (en) | 1985-09-23 | 1987-06-30 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Target specific cross-linked heteroantibodies |
IL85035A0 (en) | 1987-01-08 | 1988-06-30 | Int Genetic Eng | Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same |
WO1988007089A1 (en) | 1987-03-18 | 1988-09-22 | Medical Research Council | Altered antibodies |
DE3920358A1 (en) | 1989-06-22 | 1991-01-17 | Behringwerke Ag | BISPECIFIC AND OLIGO-SPECIFIC, MONO- AND OLIGOVALENT ANTI-BODY CONSTRUCTS, THEIR PRODUCTION AND USE |
US5208020A (en) | 1989-10-25 | 1993-05-04 | Immunogen Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
US5959177A (en) | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
US5571894A (en) | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
DK0590058T3 (en) | 1991-06-14 | 2004-03-29 | Genentech Inc | Humanized heregulin antibody |
GB9114948D0 (en) | 1991-07-11 | 1991-08-28 | Pfizer Ltd | Process for preparing sertraline intermediates |
WO1993006217A1 (en) | 1991-09-19 | 1993-04-01 | Genentech, Inc. | EXPRESSION IN E. COLI OF ANTIBODY FRAGMENTS HAVING AT LEAST A CYSTEINE PRESENT AS A FREE THIOL, USE FOR THE PRODUCTION OF BIFUNCTIONAL F(ab')2 ANTIBODIES |
US5587458A (en) | 1991-10-07 | 1996-12-24 | Aronex Pharmaceuticals, Inc. | Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof |
EP0625200B1 (en) | 1992-02-06 | 2005-05-11 | Chiron Corporation | Biosynthetic binding protein for cancer marker |
DE69233803D1 (en) | 1992-10-28 | 2011-03-31 | Genentech Inc | Use of vascular endothelial growth factor antagonists |
ATE196606T1 (en) | 1992-11-13 | 2000-10-15 | Idec Pharma Corp | THERAPEUTIC USE OF CHIMERIC AND LABELED ANTIBODIES DIRECTED AGAINST A DIFFERENTIATION ANTIGEN WHICH EXPRESSION IS RESTRICTED TO HUMAN B LYMPHOCYTES, FOR THE TREATMENT OF B-CELL LYMPHOMA |
EP0714409A1 (en) | 1993-06-16 | 1996-06-05 | Celltech Therapeutics Limited | Antibodies |
US5789199A (en) | 1994-11-03 | 1998-08-04 | Genentech, Inc. | Process for bacterial production of polypeptides |
US5731168A (en) | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
US5840523A (en) | 1995-03-01 | 1998-11-24 | Genetech, Inc. | Methods and compositions for secretion of heterologous polypeptides |
US5869046A (en) | 1995-04-14 | 1999-02-09 | Genentech, Inc. | Altered polypeptides with increased half-life |
US6267958B1 (en) | 1995-07-27 | 2001-07-31 | Genentech, Inc. | Protein formulation |
GB9603256D0 (en) | 1996-02-16 | 1996-04-17 | Wellcome Found | Antibodies |
AU743758B2 (en) | 1997-04-07 | 2002-02-07 | Genentech Inc. | Anti-VEGF antibodies |
EP0971959B1 (en) | 1997-04-07 | 2005-12-28 | Genentech, Inc. | Humanized antibodies and methods for forming humanized antibodies |
ES2246069T3 (en) | 1997-05-02 | 2006-02-01 | Genentech, Inc. | PROCEDURE FOR THE PREPARATION OF MULTI-SPECIFIC ANTIBODIES THAT HAVE COMMON AND MULTIMERIC COMPONENTS. |
US6171586B1 (en) | 1997-06-13 | 2001-01-09 | Genentech, Inc. | Antibody formulation |
EP0994903B1 (en) | 1997-06-24 | 2005-05-25 | Genentech, Inc. | Methods and compositions for galactosylated glycoproteins |
US6040498A (en) | 1998-08-11 | 2000-03-21 | North Caroline State University | Genetically engineered duckweed |
WO1999022764A1 (en) | 1997-10-31 | 1999-05-14 | Genentech, Inc. | Methods and compositions comprising glycoprotein glycoforms |
IL138608A0 (en) | 1998-04-02 | 2001-10-31 | Genentech Inc | Antibody variants and fragments thereof |
US6194551B1 (en) | 1998-04-02 | 2001-02-27 | Genentech, Inc. | Polypeptide variants |
WO1999054342A1 (en) | 1998-04-20 | 1999-10-28 | Pablo Umana | Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity |
AU1687500A (en) | 1998-12-16 | 2000-07-03 | Kyowa Hakko Kogyo Co. Ltd. | Antihuman vegf monoclonal antibody |
US6737056B1 (en) | 1999-01-15 | 2004-05-18 | Genentech, Inc. | Polypeptide variants with altered effector function |
PL209786B1 (en) | 1999-01-15 | 2011-10-31 | Genentech Inc | Variant of mother polypeptide containing Fc region, polypeptide containing variant of Fc region with altered affinity of Fc gamma receptor binding (Fc R), polypeptide containing variant of Fc region with altered affinity of Fc gamma neonatal receptor binding (Fc Rn), composition, isolated nucleic acid, vector, host cell, method for obtaining polypeptide variant, the use thereof and method for obtaining region Fc variant |
WO2000046251A2 (en) | 1999-02-05 | 2000-08-10 | Buelow Jens Ulrich | Human polyclonal antibodies from transgenic nonhuman animals |
JP4926320B2 (en) * | 1999-04-28 | 2012-05-09 | ボード・オブ・リージエンツ,ザ・ユニバーシテイ・オブ・テキサス・システム | Compositions and methods for cancer treatment by selective inhibition of VEGF |
ATE303445T1 (en) | 1999-10-04 | 2005-09-15 | Medicago Inc | METHOD FOR REGULATION OF TRANSCRIPTION OF FOREIGN GENES IN THE PRESENCE OF NITROGEN |
US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
CN101289511A (en) | 2000-04-11 | 2008-10-22 | 杰南技术公司 | Multivalent antibodies and uses therefore |
AU8470301A (en) | 2000-08-03 | 2002-02-18 | Wim-Van Schooten | Production of humanized antibodies in transgenic animals |
AU2002339845B2 (en) | 2001-08-03 | 2009-03-26 | Roche Glycart Ag | Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity |
MXPA04003798A (en) | 2001-10-25 | 2004-07-30 | Genentech Inc | Glycoprotein compositions. |
US20040093621A1 (en) | 2001-12-25 | 2004-05-13 | Kyowa Hakko Kogyo Co., Ltd | Antibody composition which specifically binds to CD20 |
WO2003085118A1 (en) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing antibody composition |
AU2003236019A1 (en) | 2002-04-09 | 2003-10-20 | Kyowa Hakko Kirin Co., Ltd. | Drug containing antibody composition appropriate for patient suffering from Fc Gamma RIIIa polymorphism |
ATE503829T1 (en) | 2002-04-09 | 2011-04-15 | Kyowa Hakko Kirin Co Ltd | CELL WITH REDUCED OR DELETED ACTIVITY OF A PROTEIN INVOLVED IN GDP-FUCOSE TRANSPORT |
EA200401325A1 (en) | 2002-04-09 | 2005-04-28 | Киова Хакко Когио Ко., Лтд. | CELLS WITH MODIFIED GENOM |
CA2481658A1 (en) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Method of enhancing of binding activity of antibody composition to fcy receptor iiia |
US7361740B2 (en) | 2002-10-15 | 2008-04-22 | Pdl Biopharma, Inc. | Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis |
US20040101920A1 (en) | 2002-11-01 | 2004-05-27 | Czeslaw Radziejewski | Modification assisted profiling (MAP) methodology |
SI1572744T1 (en) | 2002-12-16 | 2010-09-30 | Genentech Inc | Immunoglobulin variants and uses thereof |
KR101498588B1 (en) | 2003-01-22 | 2015-03-05 | 로슈 글리카트 아게 | FUSION CONSTRUCTS AND USE OF SAME TO PRODUCE ANTIBODIES WITH INCREASED Fc RECEPTOR BINDING AFFINITY AND EFFECTOR FUNCTION |
US7871607B2 (en) | 2003-03-05 | 2011-01-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases |
US20060104968A1 (en) | 2003-03-05 | 2006-05-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases |
NZ621449A (en) | 2003-05-30 | 2015-07-31 | Genentech Inc | Treatment with anti-vegf antibodies |
CA2532117C (en) | 2003-07-15 | 2012-07-10 | Therapeutic Human Polyclonals, Inc. | Humanized immunoglobulin loci |
DK1737891T3 (en) | 2004-04-13 | 2013-03-25 | Hoffmann La Roche | ANTI-P-selectin ANTIBODIES |
TWI380996B (en) | 2004-09-17 | 2013-01-01 | Hoffmann La Roche | Anti-ox40l antibodies |
KR101270829B1 (en) | 2004-09-23 | 2013-06-07 | 제넨테크, 인크. | Cystein engineered antibodies and conjugates |
JO3000B1 (en) | 2004-10-20 | 2016-09-05 | Genentech Inc | Antibody Formulations. |
CN101084317A (en) | 2004-10-22 | 2007-12-05 | 人类多克隆治疗公司 | Suppression of endogenous immunoglobulin expression |
CN103981190A (en) | 2005-02-07 | 2014-08-13 | 罗氏格黎卡特股份公司 | Antigen binding molecules that bind EGFR, vectors encoding same, and uses thereof |
DE602006015653D1 (en) | 2005-08-03 | 2010-09-02 | Therapeutic Human Polyclonals | SUPPRESSION OF B-CELL APOPTOSIS IN TRANSGENIC ANIMALS EXPRESSING HUMANIZED IMMOBILOBULINS |
US20080044455A1 (en) | 2006-08-21 | 2008-02-21 | Chaim Welczer | Tonsillitus Treatment |
CN102719444B (en) | 2006-09-01 | 2016-12-14 | 人类多细胞株治疗学公司 | The expression that people or Humanized immunoglobulin strengthen in non-human transgenic animal |
MX2009004027A (en) | 2006-10-20 | 2009-09-28 | Schering Corp | Fully human anti-vegf antibodies and methods of using. |
DE102007001370A1 (en) | 2007-01-09 | 2008-07-10 | Curevac Gmbh | RNA-encoded antibodies |
WO2009060198A1 (en) | 2007-11-09 | 2009-05-14 | Peregrine Pharmaceuticals, Inc. | Anti-vegf antibody compositions and methods |
US9266967B2 (en) | 2007-12-21 | 2016-02-23 | Hoffmann-La Roche, Inc. | Bivalent, bispecific antibodies |
US8242247B2 (en) | 2007-12-21 | 2012-08-14 | Hoffmann-La Roche Inc. | Bivalent, bispecific antibodies |
US20090162359A1 (en) | 2007-12-21 | 2009-06-25 | Christian Klein | Bivalent, bispecific antibodies |
PL2235064T3 (en) | 2008-01-07 | 2016-06-30 | Amgen Inc | Method for making antibody fc-heterodimeric molecules using electrostatic steering effects |
CA3020290A1 (en) * | 2008-06-25 | 2009-12-30 | Esbatech, An Alcon Biomedical Research Unit Llc | Stable and soluble antibodies inhibiting vegf |
WO2010112193A1 (en) | 2009-04-02 | 2010-10-07 | Roche Glycart Ag | Multispecific antibodies comprising full length antibodies and single chain fab fragments |
PT2417156E (en) | 2009-04-07 | 2015-04-29 | Roche Glycart Ag | Trivalent, bispecific antibodies |
AU2010252284A1 (en) | 2009-05-27 | 2011-11-17 | F. Hoffmann-La Roche Ag | Tri- or tetraspecific antibodies |
US9676845B2 (en) | 2009-06-16 | 2017-06-13 | Hoffmann-La Roche, Inc. | Bispecific antigen binding proteins |
CN102711809B (en) | 2009-08-17 | 2015-09-30 | 特雷康制药公司 | Use anti-endoglin antibody and anti-VEGF agent therapeutic alliance cancer |
SG179196A1 (en) | 2009-09-16 | 2012-04-27 | Genentech Inc | Coiled coil and/or tether containing protein complexes and uses thereof |
JO3340B1 (en) * | 2010-05-26 | 2019-03-13 | Regeneron Pharma | Antibodies to human gdf8 |
WO2011156328A1 (en) | 2010-06-08 | 2011-12-15 | Genentech, Inc. | Cysteine engineered antibodies and conjugates |
EP2579895A4 (en) * | 2010-06-14 | 2013-12-18 | Vaccinex Inc | Anti-vegf antibodies and uses thereof |
CU23895B1 (en) * | 2010-12-28 | 2013-05-31 | Biorec Sa | RECOMBINANT ANTIBODIES AGAINST THE GROWTH FACTOR OF THE VASCULAR ENDOTHELIUM (VEGF) OBTAINED BY MUTAGENESIS OF VARIABLE REGIONS |
ES2692268T3 (en) | 2011-03-29 | 2018-12-03 | Roche Glycart Ag | Antibody Fc variants |
EP2748202B1 (en) | 2011-08-23 | 2018-07-04 | Roche Glycart AG | Bispecific antigen binding molecules |
WO2013120929A1 (en) | 2012-02-15 | 2013-08-22 | F. Hoffmann-La Roche Ag | Fc-receptor based affinity chromatography |
US20140079691A1 (en) * | 2012-09-20 | 2014-03-20 | Anaptysbio, Inc. | Thermostable antibody framework regions |
CN103012589B (en) * | 2012-12-11 | 2014-07-16 | 上海赛伦生物技术有限公司 | Human anti-vascular endothelial cell growth factor antibody and application thereof |
WO2014177460A1 (en) | 2013-04-29 | 2014-11-06 | F. Hoffmann-La Roche Ag | Human fcrn-binding modified antibodies and methods of use |
KR101541478B1 (en) * | 2013-05-31 | 2015-08-05 | 동아쏘시오홀딩스 주식회사 | Anti-vegf antibodies and pharmaceutical composition comprising same for preventing, diagnosing or treating cancer or angiogenesis-related disease |
UA117289C2 (en) | 2014-04-02 | 2018-07-10 | Ф. Хоффманн-Ля Рош Аг | Multispecific antibodies |
CN106573986A (en) | 2014-07-29 | 2017-04-19 | 豪夫迈·罗氏有限公司 | Multispecific antibodies |
CN107108724A (en) | 2014-09-12 | 2017-08-29 | 豪夫迈·罗氏有限公司 | Cysteine engineered antibody and conjugate |
CA2980189A1 (en) | 2015-04-24 | 2016-10-27 | Genentech, Inc. | Multispecific antigen-binding proteins |
WO2017053807A2 (en) * | 2015-09-23 | 2017-03-30 | Genentech, Inc. | Optimized variants of anti-vegf antibodies |
CN111511400A (en) | 2017-12-29 | 2020-08-07 | 豪夫迈·罗氏有限公司 | anti-VEGF antibodies and methods of use thereof |
-
2018
- 2018-12-21 CN CN201880083210.8A patent/CN111511400A/en active Pending
- 2018-12-21 JP JP2020536064A patent/JP2021508471A/en active Pending
- 2018-12-21 EP EP18833877.6A patent/EP3731865A1/en active Pending
- 2018-12-21 WO PCT/EP2018/086465 patent/WO2019129677A1/en unknown
- 2018-12-21 CN CN201880081350.1A patent/CN111479588A/en active Pending
- 2018-12-21 WO PCT/EP2018/086468 patent/WO2019129679A1/en unknown
- 2018-12-21 JP JP2020536066A patent/JP7436365B2/en active Active
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- 2020-06-26 US US16/912,950 patent/US11820816B2/en active Active
- 2020-06-26 US US16/912,877 patent/US20210047395A1/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007054816A2 (en) * | 2005-11-14 | 2007-05-18 | Bioren, Inc. | Antibody ultrahumanization by predicted mature cdr blasting and cohort library generation and screening |
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