WO2024006975A1 - Procédés d'humanisation d'anticorps - Google Patents

Procédés d'humanisation d'anticorps Download PDF

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WO2024006975A1
WO2024006975A1 PCT/US2023/069480 US2023069480W WO2024006975A1 WO 2024006975 A1 WO2024006975 A1 WO 2024006975A1 US 2023069480 W US2023069480 W US 2023069480W WO 2024006975 A1 WO2024006975 A1 WO 2024006975A1
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antibody
framework region
sequence
residues
amino acid
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PCT/US2023/069480
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English (en)
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JR. Stanley KRYSTEK
Akbar Nayeem
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Bristol-Myers Squibb Company
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Publication of WO2024006975A1 publication Critical patent/WO2024006975A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present application relates to methods for designing and modifying framework regions for antibody proteins, including methods for designing humanized antibody sequences.
  • New antibodies are often developed in animal models, such as mice, rats, or rabbits. However, antibodies obtained from animal models may not be tolerated by humans because they may elicit an in vivo immune response. To reduce immunogenicity, many such antibodies intended for administration to humans are “humanized,” substituting part of their amino acid sequences by human ones. Ideally, the humanized antibodies have specificity and affinity at least as good as the parent antibody, as well as stability and other properties desirable for developability of therapeutic antibodies.
  • computational methods are available for humanizing antibodies, generally the process of making an antibody obtained from one species suitable for administration to a different species is not a straightforward process that yields predictable results; the process requires much trial-and-error.
  • an antibody is obtained from an animal model and is intended for administration to humans
  • the process can involve selecting human framework regions, grafting CDRs from the original antibody (as obtained from the animal model) onto human framework regions, and then making and testing antibodies with various mutations, such as, for example, “back mutations” which are replacements of an amino acid with the amino acid that was present at a particular position in the original antibody, so as to retain or improve the antibody’s affinity and/or obtain other desirable properties.
  • human framework region (FR) sequences may be selected by identifying, in the human genome, the germlines that produce FRs most homologous to the animal FRs of the original antibody.
  • CDR-grafted antibodies require additional amino acid changes at particular framework region residues, perhaps because such residues either are conformationally important or are in direct contact with the antigen.
  • framework changes may introduce new antigenic epitopes and, if many changes are needed, the advantages of CDR grafting over chimeric antibody construction can be lost.
  • Making and testing CDR grafted antibodies and mutant versions of such antibodies is a time consuming, trial and error process.
  • determining which heavy and light chain germline sequences should be paired to form the antibody is also important, because many pairings may not work well. For example, some pairings fail to result in an antibody that can be expressed and purified.
  • the present disclosure relates in part to a process that may be used to improve antibody humanization or otherwise to create framework substitutions to an antibody that may improve its properties.
  • the process includes using a structural annotation index (SAI) that identifies framework region residues that may be critical to binding or stability of an antibody, and to evaluate compatibility between CDRs and surrounding framework regions and to identify framework region (FR) residues that can be substituted in a CDR grafted or humanized antibody, for example, to make “back mutations”.
  • SAI structural annotation index
  • FR framework region residues that can be substituted in a CDR grafted or humanized antibody, for example, to make “back mutations”.
  • the present disclosure also relates in part to a process of identifying heavy and light chain germline sequences that tend to pair well together, as well as combining these two processes together in order to improve and streamline the preparation of humanized and other modified antibodies.
  • Exemplary embodiments herein include the following:
  • Embodiment 1 A method of modifying at least one framework region of a heavy chain variable region (VH) and/or light chain variable region (VL) of an antibody, comprising: a. selecting at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, or all amino acid positions in the VH selected from Kabat residues 22, 38, 39, 45, 46, 47, 66, 71, 73, 74, 75, 76, 86, 91, 92, 93, 94, and 103 and/or at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or all amino acid positions in the VL selected from Kabat residues 23, 36, 38, 48, 49, 61,
  • Embodiment 2 The method of embodiment 1, wherein at least one germline and/or second antibody framework region for preparation of a modified antibody is identified based on: a. percent sequence identity between the Kabat residues selected for comparison; and/or b. a lack of non-conservative amino acid differences between the residues selected for comparison.
  • Embodiment 3 The method of embodiment 1 or 2, wherein all of VH positions 22, 38, 39, 45, 46, 47, 66, 71, 73, 74, 75, 76, 86, 91, 92, 93, 94, and 103 are selected for comparison.
  • Embodiment 4 The method of embodiment 1, 2, or 3, wherein all of VL positions 23, 36, 38, 48, 49, 61, 68, 69, 81, 82, 87, 88, 98, 105, and 107 are selected for comparison.
  • Embodiment 5 The method of any one of embodiments 1-4, wherein the method does not comprise determining percent sequence identity in the VH and/or VL or in the at least one framework region of the VH and/or VL between the original antibody and the germline sequence or second antibody sequence.
  • Embodiment 6 A method of modifying at least one framework region of a heavy chain variable region (VH) and/or light chain variable region (VL) of an antibody, comprising: a. aligning an amino acid sequence of at least one framework region from an original antibody with at least one germline amino acid sequence and/or with an amino acid sequence of at least one framework region from a second antibody, and determining the precent sequence identity and/or determining location of sequence differences between the at least one framework region of the original antibody and the at least one germline sequence and/or the at least one framework region of the second antibody; b.
  • VH heavy chain variable region
  • VL light chain variable region
  • Embodiment 7 The method of embodiment 6, wherein at least one germline and/or second antibody framework region for preparation of a modified antibody is identified based on: a. percent sequence identity between the Kabat residues selected for comparison; and/or b. a lack of non-conservative amino acid differences between the residues selected for comparison.
  • Embodiment 8 The method of embodiment 6 or 7, wherein at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, or all of VH positions 22, 38, 39, 45, 46, 47, 66, 71, 73, 74, 75, 76, 86, 91, 92, 93, 94, and 103 are selected for comparison.
  • Embodiment 9 The method of embodiment 6, 7, or 8, wherein at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or all of VL positions 23, 36, 38, 48, 49, 61, 68, 69, 81, 82, 87, 88, 98, 105, and 107 are selected for comparison.
  • Embodiment 10 The method of any one of embodiments 6-9, wherein the method does not comprise determining percent sequence identity between the VH and/or VL or between the at least one framework region of the VH and/or VL.
  • Embodiment 11 A method of modifying at least one framework region of a heavy chain variable region (VH) and/or light chain variable region (VL) of an antibody, comprising: a. aligning an amino acid sequence of at least one framework region from an original antibody with at least one germline amino acid sequence and/or with an amino acid sequence of at least one framework region from a second antibody, and determining the precent sequence identity or location of sequence differences between the at least one framework region of the original antibody and the at least one germline sequence and/or the at least one framework region of the second antibody; b. determining amino acid positions in the at least one framework region from the original antibody that are not solvent exposed and do not form sheet contacts, and/or that are at the VH-VL interface; c.
  • Embodiment 12 The method of embodiment 11, wherein at least one germline and/or second antibody framework region for preparation of a modified antibody is identified based on: a. percent sequence identity between the amino acid residues determined in embodiment 11 (b); and/or b. a lack of non-conservative amino acid differences in the original antibody residues determined in embodiment 11 (b) compared to the equivalent residues of the germline sequence or second antibody sequence.
  • Embodiment 13 The method of embodiment 11 or 12, wherein the method does not comprise determining percent sequence identity in the VH and/or VL or in the at least one framework region of the VH and/or VL between the original antibody and the germline sequence or second antibody sequence.
  • Embodiment 14 A method of modifying at least one framework region of a heavy chain variable region (VH) and/or light chain variable region (VL) of an antibody, comprising: a. aligning an amino acid sequence of at least one framework region from an original antibody with at least one germline amino acid sequence and/or with an amino acid sequence of at least one framework region from a second antibody, and determining the precent sequence identity and/or determining location of sequence differences between the at least one framework region of the original antibody and the at least one germline sequence and/or the at least one framework region of the second antibody; b. assigning structural annotation index (SAI) values to the amino acid positions in the at least one framework region from the original antibody; and c. identifying at least one germline and/or second antibody framework region for preparation of a modified antibody based on the alignment of (a) and the SAI values of (b).
  • SAI structural annotation index
  • Embodiment 15 The method of embodiment 11, wherein at least one germline and/or second antibody framework region for preparation of a modified antibody is identified based on a comparison of amino acid residues with SAI values indicating that they are not solvent exposed and do not form sheet contacts and/or that are at the VH-VL interface.
  • Embodiment 16 The method of any one of embodiments 11-15, wherein at least one of Kabat residues 22, 38, 39, 45, 46, 47, 66, 71, 73, 74, 75, 76, 86, 91, 92, 93, 94, and 103 in the VH and/or at least one of Kabat residues 23, 36, 38, 48, 49, 61, 68, 69, 81, 82, 87, 88, 98, 105, and 107 in the VL are selected as residues that are not solvent exposed and do not form sheet contacts and/or that are at the VH-VL interface.
  • Embodiment 17 The method of embodiment 16, wherein at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, or all of Kabat residues 22, 38, 39, 45, 46, 47, 66, 71, 73, 74, 75, 76, 86, 91, 92, 93, 94, and 103 in the VH and/or at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or all of Kabat residues 23, 36, 38, 48, 49, 61, 68, 69, 81, 82, 87, 88, 98, 105, and 107 in the VL are selected as residues that are not solvent exposed
  • Embodiment 18 The method of any one of embodiments 1-17, wherein the method further comprises selecting at least one back mutation to be made in the modified antibody at any one or more of VH residues 22, 38, 39, 45, 46, 47, 66, 71, 73, 74, 75, 76, 86, 91, 92, 93, 94, or 103, and/or at any one or more of VL residues 23, 36, 38, 48, 49, 61, 68, 69, 81, 82, 87, 88, 98, 105, or 107.
  • Embodiment 19 The method of embodiment 18, wherein the modified antibody comprises no more than three back mutations, or no more than two back mutations, or no more than one back mutation.
  • Embodiment 20 The method of embodiment 18 or 19, wherein the at least one back mutation increases the binding affinity of the modified antibody to the antigen compared to an antibody with the same amino acid sequence but lacking the at least one back mutation.
  • Embodiment 21 The method of any one of embodiments 1-17, wherein the modified antibody comprises no back mutations.
  • Embodiment 22 The method according to any one of embodiments 14-21, wherein the method comprises assigning SAI values such that residues that are not solvent exposed and do not form sheet contacts and/or that are at the VH-VL interface that differ from residues that are solvent exposed, form sheet contacts and/or are not at the VH-VL interface.
  • Embodiment 23 The method according to any one of embodiments 14-21, wherein the method comprises determining SAI values for the at least one framework region according to Table 1.
  • Embodiment 24 The method of any one of embodiments 1-23, wherein, if the at least one framework region to be modified is in the VH, the method further comprises selecting at least one VL framework regions for pairing with the modified VH based on a database of VH/VL pairings, or wherein, if the at least one framework region to be modified is in the VL, the method further comprises selecting at least one VH framework region for pairing with the modified VL based on a database of VH/VL pairings.
  • Embodiment 25 The method of any one of embodiments 1-24, wherein the method further comprises: a. preparing a polynucleotide or a set of polynucleotides encoding the modified antibody of (c); and optionally b. preparing the modified antibody in a host cell or cell free expression system from the polynucleotide or set of polynucleotides of (d).
  • Embodiment 26 The method of embodiment 25, further comprising determining the affinity of the modified antibody for an antigen.
  • Embodiment 27 The method of any one of embodiments 1-26, wherein the original antibody is a rat, mouse, or rabbit anti-human antibody and wherein the modified antibody is a humanized antibody.
  • Embodiment 28 The method of any one of embodiments 1-26, wherein both the original antibody and the modified antibody are humanized antibodies.
  • Embodiment 29 The method of any one of embodiments 1-26, wherein the at least one framework region is compared to at least one human germline sequence in part (a).
  • Embodiment 30 The method of any one of embodiments 1-26, wherein the at least one light chain framework region is compared to a human V gene and/or J gene light chain germline sequence in part (a).
  • Embodiment 31 The method of any one of embodiments 1-26, wherein the at least one heavy chain framework region is compared to a human V gene and/or J gene heavy chain germline sequence in part (a).
  • Embodiment 32 The method of any one of embodiments 1-26, wherein the at least one light chain framework region and at least one heavy chain framework region are each compared to a human V gene and/or J gene germline sequence in part (a).
  • Embodiment 33 The method of any one of embodiments 1-32, wherein the at least one framework region comprises heavy chain framework regions FR1, FR2, and FR3.
  • Embodiment 34 The method of any one of embodiments 1-33, wherein the at least one framework region comprises light chain framework regions FR1, FR2, and FR3.
  • Embodiment 35 The method of any one of embodiments 1-34, wherein part (a) comprises performing a sequence alignment of a complete human J gene with the at least one framework region.
  • Embodiment 36 The method of any one of embodiments 1-35, wherein the method further comprises identifying complementarity determining regions (CDRs) of the original antibody.
  • CDRs complementarity determining regions
  • Embodiment 37 The method of embodiment 36, wherein the method further comprises making at least one amino acid substitution in the CDRs of the original antibody.
  • Embodiment 38 A modified antibody or a plurality of modified antibodies whose amino acid sequences are engineered using the method of any one of embodiments 1-37.
  • Embodiment 39 The modified antibody of embodiment 38, wherein the modified antibody binds to a target antigen with a dissociation constant (KD) within 10-fold or within 5-fold of the KD for binding of the original antibody to the target antigen.
  • KD dissociation constant
  • Embodiment 40 A process of determining the affinity of a modified antibody for an antigen, comprising contacting the modified antibody and the antigen under conditions in which binding affinity of antibody for antigen may be determined, wherein the modified antibody has been prepared by a method according to any one of embodiments 1-37.
  • Embodiment 41 The process of embodiment 40, wherein binding affinity is determined by surface plasmon resonance (SPR) or radioimmunoassay.
  • Embodiment 42 A system for performing the method of any one of embodiments 1- 37, wherein the system comprises computer software capable of comparing amino acid residue sequences and capable of assigning SAI values to amino acid residues.
  • Embodiment 43 The system of embodiment 42, wherein the system is further capable of identifying potential VH and VL pairings.
  • Figure 1 shows a schematic of murine, chimeric, and humanized antibodies, showing the various antibody variable and constant domains: VL (variable light chain), VH (variable heavy chain), CL (constant light), CH (constant heavy; i.e., CHI, CH2, CH3). Black lines between the shaded chains of the molecules represent natural disulfide bonds between Cys residues.
  • Figure 2 shows the arrangement of beta strands in the variable region of antibodies.
  • the different strands are labelled starting from the N-terminus (NT) and ending at the C- terminus (CT).
  • the CDRs lie on the loops joining strands B and C (CDR1), C’-C” (CDR2) and F-G (CDR3).
  • a disulfide bond connects strands B and F.
  • Figure 3 shows an exemplary flow chart for preparing humanized antibodies making use of sequence identity and the structural annotation index (SAI) described herein.
  • SAI structural annotation index
  • Figure 4 shows an exemplary table depicting a portion of a starting murine antibody light chain sequence from residue 1 to 38 (top two rows) (SEQ ID NO: 21), various possible human V gene segments (rows 9-13 (SEQ ID NO: 23-27, respectively) and their percent identity with the original murine antibody sequence (ranging from 55.21% to 59.89%), the SAI for the residues of the first framework region (row 7 and 14), followed by a comparison of the original and the chosen germline segments (rows 16-17) (SEQ ID NO: 22 and 25, respectively). CDR1 residues are shown in dark shading with white text while differences in the framework region sequences are shown in light grey shading.
  • Figures 5A-5G show binding data for murine anti-Target B antibodies and humanized variants of those antibodies, as described in Example 4 below. The data are shown as follows: mAbl (Fig. 5 A), mAb2 (Fig. 5B), mAb3 (Fig. 5C), mAb4 (Fig. 5D), mAb5 (Fig. 5E), mAb6 (Fig. 5F), and mAb7 (Fig. 5G).
  • the term “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
  • the term “about” generally refers to a range of numerical values (e.g., +/-5 to +/-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
  • the terms modify all of the values or ranges provided in the list.
  • the term about may include numerical values that are rounded to the nearest significant figure.
  • polypeptide refers to a polymer of amino acid residues, and is not limited to a minimum length.
  • a “protein” may comprise one or more polypeptides.
  • Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • a “polypeptide” or “protein” refers to a polypeptide or protein, respectively, which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site- directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification.
  • a protein may comprise two or more polypeptides.
  • antibody herein refers to a molecule comprising at least complementaritydetermining region (CDR) 1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain, wherein the molecule is capable of binding to antigen.
  • CDR complementaritydetermining region
  • the term is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, etc.), full length antibodies, single-chain antibodies, antibody conjugates, and antibody fragments, so long as they exhibit the desired specific binding activity.
  • an “isolated” antibody is one that 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
  • an “antigen” refers to the target of an antibody, i.e., the molecule to which the antibody specifically binds.
  • a “target antigen” refers to an antigen to which a particular antibody is intended to specifically bind.
  • epitope denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds.
  • Epitopes on a protein can be formed both from contiguous amino acid stretches (linear epitope) or comprise noncontiguous 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 antibody after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents.
  • heavy chain refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence.
  • a heavy chain comprises at least a portion of a heavy chain constant region.
  • full-length heavy chain refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.
  • the term “light chain” refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region.
  • the term “full-length light chain” refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.
  • 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 complementary determining regions (CDRs). See, e.g., Kindt et al. Kuby Immunology, 6 th ed., W.H. Freeman and Co., page 91 (2007).
  • a variable domain may comprise heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4; and light chain (LC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4. That is, a variable domain may lack a portion of FR1 and/or FR4 so long as it retains antigen-binding activity.
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. (See, e.g., Portolano et al., J.
  • CDRs complementarity determining regions
  • antibodies comprise six CDRs: three in the VH (CDR-H1 or heavy chain CDR1, CDR-H2, CDR-H3), and three in the VL (CDR- Ll, CDR-L2, CDR-L3).
  • CDRs herein may be defined according to the formulas of Kabat or Chothia, or another formula as described herein. (See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
  • “Framework Region” or “FR” refers to the residues of the variable region that are not part of the complementary determining regions (CDRs).
  • the FR of a variable region generally consists of four FRs: FR1, FR2, FR3, and FR4.
  • the VH and VL CDR and FR sequences generally appear in the following sequence: FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4.
  • each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CHI, CH2, and CH3).
  • VH variable domain
  • CHI variable heavy domain
  • CH2 constant heavy domain
  • VL variable domain
  • CL constant light
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain at Gly446 and Lys447 (EU numbering).
  • Antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine and lysine, respectively. Therefore, the C-terminal lysine, or the C-terminal glycine and lysine, of the Fc region may or may not be present.
  • a “full-length heavy chain constant region” or a “full length antibody” for example, which is a human IgGl antibody includes an IgGl with both a C-terminal glycine and lysine, without the C-terminal lysine, or without both the C-terminal glycine and lysine.
  • numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • Antibody effector functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
  • the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the antibody is of the IgGl isotype.
  • the antibody is of the IgGl isotype with the L234A, L235A, and P329G (LALAPG) mutation to reduce Fc-region effector function.
  • the antibody is of the IgG2 isotype.
  • the antibody is of the IgG4 isotype with the S228P mutation in the hinge region to improve stability of IgG4 antibody.
  • the antibody may have a non-human IgG constant region, and may be, for example, a murine IgG2a antibody such as a murine IgG2a LALAPG antibody.
  • the heavy chain constant domains that correspond to the classes IgA, IgD, IgE, IgG, and IgM are called a, 5, £, y, and
  • the light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.
  • 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.
  • 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 scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments.
  • full length antibody “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or, in the case of an IgG antibody, having heavy chains that contain an Fc region as defined herein above.
  • An antibody that is “modified” as compared to an “original” (i.e. starting) antibody comprises at least one amino acid substitution, deletion or insertion compared to the original antibody.
  • the modified antibody comprises modifications in the framework regions.
  • a “CDR grafted antibody,” as used herein, refers to an antibody comprising the CDRs of the original antibody and framework regions derived from a different source, such as one or more V, D, or J germline sequences, or from a different antibody.
  • the CDRs of an antibody can be combined with framework regions from either a germline sequence or a different antibody.
  • a CDR grafted antibody is a chimeric antibody, such as a humanized antibody, in which the CDRs of, for instance, a murine or rabbit or rat antibody are grafted onto germline sequences from a different species, such as a human, which provide the surrounding framework sequences.
  • a CDR grafted antibody replaces the framework regions of an existing antibody with framework regions of a different antibody, for example, or with framework regions derived from a different germline sequence from the same animal species.
  • a CDR grafted antibody in order to change the framework regions from those of one human germline sequence to those of another human germline sequence.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs; a “murinized” antibody refers to a chimeric antibody comprising residues from non-murine CDRs and amino acid residues from murine FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody refers to an antibody that has undergone humanization.
  • a humanized antibody may comprise a CDR grafted antibody as well as a CDR grafted antibody that comprises further amino acid changes in the framework region or in the CDRs, for example, in order to improve the properties of the humanized antibody.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • a “multispecific” antibody is one that binds specifically to more than one target antigen, while a “bispecific” antibody is one that binds specifically to two antigens.
  • An “antibody conjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a therapeutic agent or a label.
  • Percent (%) amino acid sequence identity and “percent identity” and “percent homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • leader sequence refers to a sequence of amino acid residues located at the N terminus of a polypeptide that facilitates secretion of a polypeptide from a mammalian cell.
  • a leader sequence may be cleaved upon export of the polypeptide from the mammalian cell, forming a mature protein.
  • Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are attached. Nonlimiting exemplary leader sequences also include leader sequences from heterologous proteins.
  • an antibody lacks a leader sequence.
  • an antibody comprises at least one leader sequence, which may be selected from native antibody leader sequences and heterologous leader sequences.
  • nucleic acid molecule or “polynucleotide” includes any compound and/or substance that comprises a polymer of nucleotides.
  • Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine I, guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group.
  • cytosine I guanine
  • A adenine
  • T thymine
  • U uracil
  • sugar i.e. deoxyribose or ribose
  • phosphate group i.e. deoxyribose or ribose
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules.
  • DNA deoxyribonucleic acid
  • cDNA complementary DNA
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • the nucleic acid molecule may be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single stranded and double stranded forms.
  • nucleic acid molecule can contain naturally occurring or non- naturally occurring nucleotides.
  • non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues.
  • Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and/or in vivo, e.g., in a host or patient.
  • DNA e.g., cDNA
  • RNA e.g., mRNA
  • 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.
  • 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 selfreplicating 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”.
  • expression vectors are referred to herein as “expression vectors”.
  • 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.
  • binding or “binding” or “specific binding” and similar terms, when referring to a protein and its ligand or an antibody and its antigen target for example, or some other binding pair, means that the binding affinity between the members of the binding pair is sufficiently strong that the interaction cannot be due to random molecular associations (i.e. “nonspecific binding”).
  • 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).
  • binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • Affinity can generally be represented by the dissociation constant (KD).
  • KD dissociation constant
  • Affinity of an antibody for an antigen can be measured by common methods known in the art, such as surface plasmon resonance (SPR), for instance.
  • the present disclosure includes, for example, methods of making and testing antibodies with modified framework regions, as well as systems for carrying out the methods, the modified antibodies, nucleic acids encoding them, and associated vectors and host cells.
  • the modified antibodies are CDR grafted antibodies, such as chimeric antibodies (e.g., humanized or murinized antibodies) wherein the framework regions are derived from germlines from a species different from the original antibody (e.g., rat antihuman or human anti-mouse, etc.).
  • Antibodies herein are broadly interpreted, and encompass molecules comprising at least CDR1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain, wherein the molecules are capable of binding to antigen.
  • antibodies herein may contain heavy and light chain segments comprising CDR1-FR2- CDR2-FR3-CDR3 and at least a portion of FR1 and/or FR4 of the VH and VL, either prepared or expressed as separate VH and VL polypeptide chains or alternatively, prepared or expressed as a single polypeptide chain (i.e., a single-chain antibody optionally with a linker in between the polypeptide chains).
  • antigen binding structures include but not limited to antigen binding fragments, such as Fab, Fab’, Fab’-SH, Fv, scFv, or F(ab’)2 fragments, antibodies comprising an Fc domain, full length antibodies, single-chain antibodies, antibodies of various classes, such as IgG, IgM, IgA, and the like, multispecific antibodies (e.g., bispecific antibodies, diabodies, etc.), and antibody conjugates, so long as they comprise a heavy chain variable region (VH) and a light chain variable region (VL) comprising the CDRs and the surrounding framework regions that may be swapped or modified according to methods herein.
  • VH heavy chain variable region
  • VL light chain variable region
  • the modified antibody is an antibody fragment, such as a Fab, Fab’, Fv, scFv, or F(ab’)2 fragment.
  • Papain cleaves antibodies above the so-called hinge region that joins two sets of heavy and light chains together. Papain cleavage produces two identical “Fab” fragments, each containing a VH-CH1 and a VL-CL bound together, as well as an Fc fragment, for example, comprising the remaining heavy chain constant region segments.
  • “Fab’ fragments” in contrast to Fab fragments contain further residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region.
  • an F(ab')2 fragment which comprises two Fab fragments and the hinge region that joins the two fragments.
  • An Fv fragment comprises only the VH and VL segments of a Fab, without the CHI or CL segments.
  • An scFv is an Fv molecule that is made in a continuous single strand, for example, by adding a flexible linker peptide in between the VH and VL portions of the peptide. Variations on each of these fragments are also encompassed herein, so long as they contain the 6 antibody CDRs and the intervening framework regions, which are to be modified in methods herein. Examples include diabodies or triabodies, which are constructs of 2 or more Fv fragments, for instance.
  • antibodies herein are full length antibodies.
  • Methods herein apply to antibodies regardless of their target antigen or epitope and regardless of their degree of affinity for such an antigen or epitope.
  • methods herein include first assigning the CDRs of an antibody to be modified.
  • the original antibody may be a murine anti-human antibody to be humanized according to methods herein, or a humanized antibody whose framework regions are to be re-engineered based on a different human germline sequence.
  • CDR assignment may be done according to prior methods.
  • Kabat CDRs or modified Kabat CDRs may be assigned according to the Kabat and related numbering scheme.
  • one of the following numbering formats may be used, as shown in the table below, for example.
  • the table below uses Kabat residue numbering for the amino acid residue positions as a whole. (Table A below is based on www.bioinf. org.uk/abs/info. html#kabatnum.)
  • the Kabat CDR assignment in the table below is used.
  • the Chothia assignment is used.
  • the Kabat CDR assignment in the table below is used for all CDRs except CDRH1, and that the CDRH1 is assigned according to the Chothia definition.
  • the AbM assignment is used.
  • the Contact assignment is used.
  • IMGT assignment is used.
  • CDR-L1 starts at residue 24, which follows a Cys at residue 23, and the CDR proceeds for 10-17 residues and is followed by a Trp, typically in a Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln, or Trp-Tyr-Leu stretch.
  • Trp typically in a Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln, or Trp-Tyr-Leu stretch.
  • Trp typically in a Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln, or Trp-Tyr-Leu stretch.
  • Trp typically in a Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln, or Trp-Tyr-Leu stretch.
  • CDR-L2 starts 16 residues after the end of CDR
  • CDR-L3 starts 33 residues after the end of CDR-L2, the residue prior to the CDR is a Cys, and the residues that follow it are Phe-Gly-Xxx-Gly, and its length is 7-11 residues.
  • CDR-H1 starts at residue 26, which is located 5 residues after a Cys, while the residue that follows the CDR is a Trp, often Trp-Val, Trp-Ala, or Trp-Ile.
  • CDR-H2 is 15 residues after the end of CDR-H1 and can be 16-19 residues long.
  • CDR-H3 is usually 33 (but sometimes 30) residues after the end of CDR-H2 and always 3 residues after a Cys.
  • the residues that follow the CDR are generally Trp-Gly-Xxx-Gly.
  • the CDR can be anywhere from 3 to about 25 residues in length. Similar rules for assigning CDRs according to Chothia (See Al-Lazikani B, Lesk AM, Chothia C, J Mol Biol. 1997 Nov 7; 273(4):927- 48) and other CDR definitions are described in the literature.
  • a modified antibody such as a CDR grafted antibody
  • alignment may further comprise determining the percent sequence identity of at least one framework region of the alignment (i.e., FR1, FR2, FR3, or FR4) or of all of the framework regions, or of a stretch including at least one framework region and at least part of the CDRs.
  • V gene segments are available from various sources, for example, the VBASE2 database (I. Retter et al., Nucl. Acids Res. 33(Suppl. 1) D671-D674 (2005)), Kabat database (G. Johnson & T.T. Wu, Nucl. Acids Res. 29: 205-206 (2001)), IMGT database (M.P. Lefranc, Methods Mol. Biol. 248: 27-49 (2004), among others. Sequences and alignments of V, D, and J gene segments are also available, for example, at https://www2.mrc- lmb.cam.ac.uk/vbase/alighments2.php, among other locations.
  • the 20 amino acids of the genetic code may be grouped based on chemical properties of their side chains, such as, hydrophobic (e.g., Met, Ala, Vai, He, Leu), hydrophilic (Cys, Ser, Thr, Asn, Gin), acidic (Asp, Glu), basic (His, Lys, Arg), and aromatic (Trp, Phe, Tyr), and residues that can affect the peptide backbone orientation (Gly, Pro).
  • side chains such as, hydrophobic (e.g., Met, Ala, Vai, He, Leu), hydrophilic (Cys, Ser, Thr, Asn, Gin), acidic (Asp, Glu), basic (His, Lys, Arg), and aromatic (Trp, Phe, Tyr), and residues that can affect the peptide backbone orientation (Gly, Pro).
  • conservative substitutions of one amino acid for another retain such side-chain properties. In some cases, conservative substitutions also retain the shape or size of the original residue.
  • non-conservative substitutions which may impact the properties of a side chain include substitution of an acidic residue (Asp, Glu) for a basic residue (His, Lys, Arg) and vice versa or substitution of a small side chain such as Gly, Ala, Vai, He, Leu with that of a large aromatic side chain such as Trp, Tyr, or Phe, for example.
  • the structural annotation index may identify framework amino acid residue positions in which substitutions are more and less likely to be tolerated and to impact antigen binding.
  • the SAI provides further information about the structural role of particular framework residues, and accordingly, can be used to make better choices among human germline segments for humanization, for example, and to improve the selection of back mutations and other amino acid changes after CDR grafting to be tested.
  • the structural annotation index was developed to provide additional information to assist selection of an appropriate human germline sequence for humanization of a heavy chain and/or a light chain of an antibody.
  • the SAI helps to identify exposed side chains pointing outwards into solvent, which may be readily altered during humanization, compared to buried side chains that point into the core of the antibody and side chains that could impact antigen binding either directly or indirectly.
  • the SAI positions were developed based on high resolution X-ray crystal structures of several antibodies and associated testing, as described in Table 1 and in the Examples herein. As shown in Table 1, each non-CDR VH or VL position is assigned a number, with some numbers also being italicized and others in plain text.
  • Italicized numbers in Table 1 indicate relatively important residue types, in which a substitution, particularly a non-conservative substitution, might be more likely to impact antigen binding interactions.
  • the italicized positions of Table 1 represent residues that are not solvent exposed and do not form sheet contacts (i.e., contacts that form the beta sheets of the framework region), or residues that are at the VH-VL interface.
  • these residues generally tend to be close to the CDRs or make interactions with CDR residues, could impact VH-VL interactions, or otherwise could impact antigen binding interactions by being close to the antigen-antibody interface.
  • these italicized SAI positions tend to be located at residues 22, 38, 39, 45, 46, 47, 66, 71, 73, 74, 75, 76, 86, 91, 92, 93, 94, and 103 of the VH (Kabat numbering) and at residues 23, 36, 38, 48, 49, 61, 68, 69, 81, 82, 87, 88, 98, 105, and 107 of the VL (Kabat numbering).
  • the SAI information can be evaluated solely or along with percent identity information for the entire framework region being considered in order to select a suitable human germline sequence for the humanization of antibodies.
  • the SAI information may also be combined with information about preferred pairings of particular VH and VL germline segments, e.g. from a statistical database as described herein, in order to select particular germlines for humanization of VH and VL segments and to design any amino acid substitutions after CDR grafting.
  • the SAI may also help to direct the design of framework region substitutions so as to restore high affinity binding to an antigen after CDR grafting or even further enhance antigen binding compared to a parental non-human antibody.
  • a suitable set of heavy and/or light chain germline sequences to serve as the basis for the CDR grafting may be selected in part through performing alignments between original and potential framework regions and/or noting residue differences in an alignment of an original and a potential substitute framework region either from a germline gene or from a different antibody.
  • the same process may be used for designing other types of modified antibodies in which the framework regions are modified.
  • the SAI provides yet further information that may be used to select an appropriate germline sequence for further antibody engineering, by identifying specific residues within a heavy chain or light chain framework segment, for example, that may readily tolerate amino acid residue changes compared to those that may not tolerate sequence changes easily.
  • the SAI not only provides further information for the germline selection process, but may also guide further engineering of antibody framework regions, for example, by identifying locations where a “back mutation” (i.e., a substitution of a residue in a new human germline framework sequence back to the original residue from the original murine framework sequence, for example) may be beneficial.
  • a “back mutation” i.e., a substitution of a residue in a new human germline framework sequence back to the original residue from the original murine framework sequence, for example
  • certain framework residues may, for example, make interactions with residues of a nearby CDR, or may be located at particular interfaces between a VH and VL or may be close to the antigen-antibody interface.
  • Sequence changes in these residues in a CDR grafted antibody are expected to be more likely to disrupt antigen binding or VH/VL interaction than sequence changes at other locations in the framework region. This information can assist in choosing an appropriate germline sequence (i.e. one with fewer sequence changes at those locations compared to the original antibody to be modified than other possible germline sequences) and/or in designing appropriate mutations (e.g., back mutations) to make in a CDR grafted antibody.
  • methods provided herein may comprise: methods of modifying at least one VH framework region of an antibody, comprising: selecting at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, or all eighteen of the amino acid positions in the VH selected from Kabat residues 22, 38, 39, 45, 46, 47, 66, 71, 73, 74, 75, 76, 86, 91, 92, 93, 94, and 103 and/or modifying at least one VL framework region of an antibody comprising: selecting at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or all fifteen of the amino acid positions in the VH selected from Kabat residue
  • Methods herein also include methods of modifying at least one framework region of a heavy chain variable region (VH) and/or light chain variable region (VL) of an antibody, comprising: aligning an amino acid sequence of at least one framework region from an original antibody with at least one germline amino acid sequence and/or with an amino acid sequence of at least one framework region from a second antibody, and determining the precent sequence identity and/or determining the location of sequence differences between the at least one framework region of the original antibody and the at least one germline sequence and/or the at least one framework region of the second antibody; determining whether sequence differences between the at least one framework region of the original antibody and the at least one germline sequence and/or the at least one framework region of the second antibody occur at any one or more of Kabat residues 22, 38, 39, 45, 46, 47, 66, 71, 73, 74, 75, 76, 86, 91, 92, 93, 94, and 103 in the VH (e.g., at least two, at least two, at least
  • a germline and/or second antibody framework region to be used for preparation of a modified CDR grafted antibody can then be selected based solely on the percent sequence identity between the Kabat residues selected for comparison; and/or a lack of non-conservative amino acid differences between the residues listed above that have been selected for comparison.
  • additional factors may be used to choose the germline and/or second antibody framework region, such as the percent sequence identity between the original framework region and that of the germline or second antibody, and a lower extent of non-conservative substitutions between the original framework and that of the germline or second antibody, as well as information from a database of preferred VH and VL pairings, as discussed further below.
  • methods herein include determining percent sequence identity in the VH and/or VL or in the at least one framework region of the VH and/or VL between the original antibody and the germline sequence or second antibody sequence. In other cases, this step is not included. Thus, in some cases, the methods do not comprise determining percent sequence identity between the VH and/or VL or between the at least one framework region of the VH and/or VL.
  • Further methods herein include, for example, modifying at least one framework region of a heavy chain variable region (VH) and/or light chain variable region (VL) of an antibody, comprising: (a) aligning an amino acid sequence of at least one framework region from an original antibody with at least one germline amino acid sequence and/or with an amino acid sequence of at least one framework region from a second antibody, and determining the precent sequence identity or location of sequence differences between the at least one framework region of the original antibody and the at least one germline sequence and/or the at least one framework region of the second antibody; determining amino acid positions in the at least one framework region from the original antibody that are not solvent exposed and do not form sheet contacts, and/or that are at the VH-VL interface; comparing the amino acid residues at the positions determined in (b) with the amino acid residues found at the equivalent positions of a VH and/or VL from at least one germline and/or at least one second antibody; and identifying at least one modified antibody framework sequence based on the comparison of (c
  • At least one germline and/or second antibody framework region for preparation of a modified antibody is identified based on percent sequence identity between the amino acid residues of the original set of amino acid residues of (b); and/or a lack of nonconservative amino acid differences in the original antibody residues of (b) compared to the equivalent residues of the germline sequence or second antibody sequence.
  • such a method does not comprise determining percent sequence identity in the VH and/or VL or in the at least one framework region of the VH and/or VL between the original antibody and the germline sequence or second antibody sequence.
  • Yet further methods of modifying at least one framework region of a heavy chain variable region (VH) and/or light chain variable region (VL) of an antibody comprise: (a) aligning an amino acid sequence of at least one framework region from an original antibody with at least one germline amino acid sequence and/or with an amino acid sequence of at least one framework region from a second antibody, and determining the precent sequence identity and/or determining location of sequence differences between the at least one framework region of the original antibody and the at least one germline sequence and/or the at least one framework region of the second antibody; (b) assigning structural annotation index (SAI) values to the amino acid positions in the at least one framework region from the original antibody; and (c) identifying at least one germline and/or second antibody framework region for preparation of a modified antibody based on the alignment of (a) and the SAI values of (b).
  • SAI structural annotation index
  • At least one germline and/or second antibody framework region for preparation of a modified antibody is identified based on a comparison of amino acid residues with SAI values indicating that they are not solvent exposed and do not form sheet contacts and/or that are at the VH-VL interface.
  • the SAI values may be assigned in various ways. For example, in some cases, one may only distinguish between residues that fall into the italicized categories of Table 1 of the Examples below from residues that are not in the italicized categories. In such a case, there would only be two different SAI values, and any type of denomination could be used to distinguish them according to preference. In other cases, more than two SAI categories could be used, for example, to call out one or two types of residue classes from the others, such as solvent exposed vs. all others, or residues that are solvent exposed or that make sheet contacts vs.
  • a computer software program may be used to assign SAI values to particular residues and to keep track of multiple parameters affecting the choice of a germline or second antibody sequence as the basis for preparing a modified antibody.
  • the disclosure also includes appropriate software systems for use in conducting the methods herein.
  • a software system could be used to summarize percent identity, preferred VH-VL pairings, and SAI information to aid in the design of modified antibodies.
  • modified antibodies are CDR grafted antibodies.
  • modified antibodies comprise at least one additional amino acid modification, such as a substitution, deletion, or insertion, in the framework region.
  • that modification is a back mutation.
  • the modification is in one or more CDR regions.
  • residues that are not solvent exposed, not part of the sheet contacts, and/or that are at the VH/VL interface may be those in which a difference between an original residue and a different residue in a chosen germline or second antibody framework sequence could be most critical and thus, where a back mutation may be helpful.
  • methods of designing modified antibodies herein also include designing particular back mutations at residues whose SAI values indicate that such back mutations may be helpful in restoring higher binding affinity, for example.
  • no more than one back mutation in the VH and/or no more than one back mutation in the VL is ultimately prepared following the sequence analysis. In other cases, no more than two back mutations in the VH and/or in the VL are prepared. In yet other cases, no more than three back mutations in the VH and/or in the VL are prepared.
  • methods herein include assigning CDRs to an original antibody framework region, using the SAI to identify the surrounding framework residues that correspond to residues potentially important for maintaining antigen binding affinity, and comparing the sequence at those residues between the original framework and a potential new framework sequence, optionally also determining overall percent sequence identity between the original and potential new framework, and optionally designing potential back mutations.
  • An example of how an SAI may be used to assist in designing modified antibodies is depicted in Figure 4 and further described in the Examples. This figure shows the first 38 residues of a light chain sequence to be humanized. These residues correspond to positions 1-36 in the alignments, as shown in rows 7, 26, and 45, for example, and Kabat positions 1-32, as shown in row 49.
  • the SAI codes for these positions are shown in rows 5, 14, 31, and 48.
  • the CDR1 residues are shown in dark shading, and correspond to SAI code 13 (rows 5, 14, 31, and 48).
  • Sequences corresponding to the segments shown in Fig. 4 are provided in the sequence table below.
  • FIG. 4 Exemplary selection of a V germline sequence.
  • the original murine variable region sequence of Fig. 4 was aligned with several possible human V kappa germline sequences, IGKV3-l l*01, IGKV3-15*01, IGKV4-l*01, IGKV1-39*O1, and IGKV2-30*01, as shown in rows 7-17 of Fig. 4, and the IGKV4-l*01 was chosen for further analysis.
  • rows 9-13 column 3, of Fig. 4 all of these possible depicted germline sequences have between 55% and 60% homology with the original antibody.
  • Each has several amino acid differences within the first framework region depicted in Fig.
  • SAI indicates that these residues are potentially solvent exposed or far from the CDRs (codes 8, 10 and 11) or are strand/sheet contacts (codes 9 and 12)
  • back mutations in those residues might not be needed in this framework region, or might be limited to mutations that correct only a non-conservative substitution, for example.
  • Fig. 4 also shows that the chosen IGKV4-l*01 germline sequence differs at position 15 from the original antibody sequence in that it has an L while the original sequence has a P.
  • row 51 of the figure shows that a possible L to P back mutation could be prepared in order to address this non-conservative amino acid change.
  • FIG. 4 further shows alignment of the C-terminal portion of the light chain variable region with various human J germline segments, and the corresponding SAI values for these residue positions. (See rows 33-44.)
  • the first, last, and third from last of these framework residue positions have SAI values of 38 or 7, indicating that they may contribute to the antibody structure and may be less tolerant to mutations.
  • each of the human J gene segments compared here contains a lysine rather than an arginine in the last position shown, which is a conservative substitution, given the potential importance of this residue according to the SAI information, it would be reasonable to consider a back mutation to an arginine at that location in designing humanized antibodies.
  • other residue substitutions may be helpful to include, either in a framework or in a CDR region, for example, to limit potential liabilities due to possible oxidation or deamidation or the like and therefore improve stability and limit degradation of the antibody during storage.
  • a spreadsheet or similar software platform e.g. Microsoft Excel® or the like
  • a platform could be used to assign CDR sequences, perform appropriate alignments, and perform the SAI analysis after alignments, for example.
  • the methods herein may also comprise obtaining their coding sequences and optionally preparing such modified antibodies for testing.
  • the present methods may include selecting a VH and VL pairing for a modified antibody. For example, if a particular VH framework region is analyzed according to methods herein and an appropriate germline VH sequence is chosen, before designing a modified antibody, one may further determine an appropriate pairing VL germline sequence to go with the chosen VH. In part, one may choose a VL by following the same process of comparing the potentially important VL framework residues of the original VL to those of potential new VL frameworks, based on the SAI information herein.
  • a modified VH is chosen for preparation, for example, one may as a further step select a VL framework sequence to pair with the modified VH in part based on pairings observed in such a database. And vice versa, once a modified VL sequence is identified, one may select a VH framework sequence to pair with it in part based on sequence pairings compiled in a database. For example, in some embodiments, such a database may be used to obtain preferred human VH-VL pairings.
  • Nucleic acid molecules comprising polynucleotides that encode one or more chains of modified antibodies herein also are provided.
  • Modified antibodies designed according to methods herein may be produced, for example, by designing a polynucleotide or set of polynucleotides for expression of the antibodies.
  • a single polynucleotide may comprise separate open reading frames encoding a light chain and a heavy chain; a single polynucleotide may comprise an open reading frame encoding a complete single chain antibody construct comprising the heavy and light chains joined by a linker; or two different polynucleotides may be used with one comprising an open reading frame encoding a light chain and the other comprising an open reading frame encoding a heavy chain.
  • Such polynucleotides may be incorporated into one or more vectors for use in a host cell or in a cell free expression system. For initial testing of potential antibodies, for example, transient transfection may be utilized to obtain relatively small quantities of an antibody species.
  • a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an antibody. In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.
  • the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides.
  • a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.
  • a polynucleotide encoding a heavy chain or light chain comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N terminus of the heavy chain or light chain.
  • the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.
  • Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art.
  • a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.
  • Vectors comprising polynucleotides that encode heavy chains and/or light chains are provided.
  • Vectors comprising polynucleotides that encode heavy chains and/or light chains are also provided.
  • Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc.
  • a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain.
  • the heavy chain and light chain are expressed from the vector as two separate polypeptides.
  • the heavy chain and light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.
  • a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain.
  • the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts).
  • a mole- or mass-ratio of between 5: 1 and 1 :5 of the first vector and the second vector is transfected into host cells.
  • a mass ratio of between 1 : 1 and 1 :5 for the vector encoding the heavy chain and the vector encoding the light chain is used.
  • a mass ratio of 1 :2 for the vector encoding the heavy chain and the vector encoding the light chain is used.
  • a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer et al., BiotechnoL Prog. 20:880-889 (2004).
  • a vector is chosen for in vivo expression of heavy chains and/or light chains in animals, including humans.
  • expression of the polypeptide is under the control of a promoter that functions in a tissuespecific manner.
  • tissuespecific promoters are described, e.g., in PCT Publication No. WO 2006/076288.
  • heavy chains and/or light chains may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art.
  • eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells;
  • PER.C6® cells Crucell
  • NSO cells anti-PAD4 heavy chains and/or anti-PAD4 light chains may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 Al.
  • a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
  • nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.
  • one or more polypeptides may be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method.
  • Antibodies may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. [00143] In some embodiments, an antibody is produced in a cell-free system.
  • Nonlimiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21 : 695-713 (2003).
  • Modified antibodies made according to methods herein may be tested in various ways to determine their properties compared to the original, unmodified antibody. For instance, CDR grafted antibodies can, in some cases, show reduced binding affinity for the target antigen. Thus, in some cases, one may determine the binding affinity of modified antibodies made based on methods herein.
  • the affinity of an antibody for an antigen can be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art, including but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics methods (e.g., surface plasmon resonance analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration).
  • in vitro assay methods biochemical or immunological based assays
  • equilibrium methods e.g., enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA)
  • kinetics methods e.g., surface plasmon resonance analysis
  • indirect binding assays e.g., competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and
  • SPR surface plasmon resonance
  • a BIACORE® surface plasmon resonance assay e.g., BIACORE®-2000 or a BIACORE ®-3000 (BIAcore, Inc., Piscataway, NJ)
  • one binding partner such as an antigen
  • CM5, BIACORE, Inc. carboxymethylated dextran biosensor chips
  • EDC N- ethyl-V- (3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N- hydroxysuccinimide
  • Such a system allows for determination of the association rates (kon) and dissociation rates (koff) of the interaction, for example, using a simple one-to-one Langmuir binding model (BIACORE ® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. Affinity may then be determined as the equilibrium dissociation constant (KD), which is calculated as the ratio of koff to kon.
  • KD equilibrium dissociation constant
  • affinity measured as a KD or IC50 can be determined by an affinity assay in which a labeled antigen, for instance, immobilized on a chip or multi-well plate, is titrated with increasing concentrations of antibody.
  • affinity assays include radioimmunoassays (RAI), ELISA, and the like, for instance.
  • a modified antibody herein may not bind the target antigen.
  • the modified antibody may have an affinity for the target antigen (i.e., a KD or IC50) that is within 10-fold of that of the original antibody (i.e. from 10-fold lower to 10-fold higher KD or IC50).
  • the affinity may be within 5-fold of that of the original antibody.
  • it may be within 2-fold of that of the original antibody.
  • the modified antibody may have a higher affinity for a target antigen than the original antibody (i.e., as measured by a lower KD or IC50 than for the original antibody).
  • modified antibodies herein comprise one or more constant regions, such as human constant regions or murine constant regions. In other embodiments, the antibodies do not comprise constant regions.
  • antibodies engineered using methods herein are also contemplated.
  • such antibodies may comprise a human heavy chain constant region of an isotype selected from IgA, IgG, and IgD.
  • the human light chain constant region is of an isotype selected from K and X.
  • an antibody described herein comprises a human IgG constant region, such as an IgGl, IgG2, IgG3, or IgG4.
  • it comprises a murine IgG constant region, such as a murine IgGl, IgG2a, or IgG2b.
  • Antibodies herein may have a variety of heavy chain constant region sequences.
  • the choice of heavy chain constant region may depend on the desired properties of the antibody.
  • a heavy chain constant region can determine whether or not an antibody will have effector function in vivo.
  • a number of point mutations can affect the degree of effector function of the antibody, for example.
  • an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the Fc region of a heavy chain constant region can comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art.
  • Another modification of the antibodies described herein is addition of a polymer such as PEG (i.e., pegylation).
  • An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
  • Examples 1-5 below and the associated figures and tables describe the development of the structure annotation index (SAI) and its use in designing modified antibodies against a murine target antigen and a human target antigen.
  • SAI structure annotation index
  • FIG. 3 A murine antibody target (Target B) was chosen for humanization. The variable regions of the heavy and light chains were compared to those of the human (Target B) germlines to find the best match. It should be noted that the selected germline was not always the one that showed the highest sequence identity to the target, but was often that which had the fewest number of amino acid differences in framework regions that were deemed critical in terms of the structure annotation index (SAI).
  • SAI structure annotation index
  • Vernier zone residues typically included the Vernier zone residues, which have been known to critically affect antigen binding by making direct contact with the antigen or by affecting the CDR loop (for a discussion of Vernier zone residue mutations and how they affect thermodynamic stability see for example, Koki Makabe, Takeshi Nakanishi, Kouhei Tsumoto, Yoshikazu Tanaka, Hidemasa Kondo, Mitsuo Umetsu, Yukiko Sone, Ryutaro Asano, and Izumi Kumagai, JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 2, pp. 1156 -1166, January 11, 2008).
  • chain-packing residues are residues buried at the VH/VL interface that could affect conformation of the antigen binding pocket. Because these residues may alter the structure of CDRs and antibody’s affinity for the antigen, the humanization procedure retained the mouse residues (back mutation) at these positions. Platform for humanization
  • the input in FASTA format was entered into the first cells of rows 1 and 2, as shown in Fig. 4.
  • the CDRs were assigned using a set of rules for determining the CDRs in an antibody sequence provided at: http://www.bioinf.org.Uk/abs/info.html#cdrdef and displayed in row 4 of Fig. 4.
  • the assignment of the first CDR of the heavy chain (CDRH1) followed the Chothia definition, which is based on the location of the structural loop rather than sequence variability (Kabat) since this was judged to be more relevant for antibody construct designs.
  • the SAI numbers at each residue position were assigned using sequence alignment with Avastin® and the preassigned SAI numbers for Avastin® as described above and displayed in row 5 of Fig. 4.
  • the five V germline sequence families with highest sequence identity to the test sequence were aligned to the test sequence (rows 8-13).
  • the corresponding sequence identities to the test sequence were calculated and displayed (rows 9-13). Differences in residues are highlighted by grey shading, and CDRs are shown in black shading with white text. Looking at residue differences in the framework regions and the SAI assignments (row 14), the germline family deemed most suitable for humanization based on sequence identity and other considerations, was selected from the five germlines in rows 9- 13.
  • the germline family selected for humanization of the light chain was IGKV4-l*-01, from row 11. This row number is entered in row 16, which then produced the sequence alignment of IGKV4-l*-01 with the test sequence.
  • a number of heavy chain germlines are shown that best pair with this germline, taken from a database of preferred VH/VL pairings (Rows 20- 25) (see Example 5).
  • Residues for back mutations were then selected for further constructs and are entered at the appropriate residue position in Row 51 and below, e.g., residue 15 (Leu) was mutated back to a Pro (Row 51) which is the original residue from the test sequence (Row 46).
  • All parental, chimeric, and humanized antibodies were produced by transient transfection of HEK293-6E cells, and the culture media was harvested 5 days after transfection.
  • concentration/titer of antibody supernatants were determined using Biolayer Interferometry (BLI) on a Fortebio® Octet RED (Rapid system — Extended Detection) biosensor instrument (Fortebio, Inc. Menlo Park, CA), by capturing antibodies from supernatant on protein A coated biosensors (Pall Fortebio #18-5010) and measuring the capture response with respect to a standard curve obtained using a control antibody sample.
  • Antibody supernatants were prepared for capture on biosensor surfaces by diluting in 10 mM sodium phosphate, 130 mM sodium chloride, 0.05% tween 20, pH 7.1 (PBS-T buffer) to 10 pg/ml for Octet BLI studies or 5 pg/ml for Biacore® SPR studies, or captured undiluted if supernatant concentration was less than these target concentrations.
  • Biacore® SPR studies were performed on a Biacore® T200 biosensor instrument (Cytiva, Marlborough, MA). Antibodies were captured on protein A coated CM5 sensor chip (Cytiva # 29127557) using a 15 second (s) contact time at 10 pl/min flow rate. This was followed by the binding of purified Target-B analyte at various concentrations, using 180 s association and dissociation times at a flow rate of 30p/min. All steps were performed in PBS-T running buffer at 25°C. SPR data were fit to a 1 : 1 Langmuir model using Biacore® T200 evaluation software to obtain values for the association rate constant (k a ), dissociation rate constant (ka) and dissociation constant (KD).
  • association rate constant k a
  • ka dissociation rate constant
  • KD dissociation constant
  • a common technique for humanization of non-human antibodies entails grafting their complementarity determining regions (CDRs) onto the variable light (VL) and variable heavy (VH) framework regions of human immunoglobulin molecules, while retaining those non-human framework residues deemed essential for the integrity of the antigen-combining site (Fig. 1).
  • CDRs complementarity determining regions
  • VL variable light
  • VH variable heavy
  • a non-human antibody such as a murine antibody
  • this entails grafting the CDRs of the non-human antibodies onto the human template.
  • human germline frameworks with highest sequence identity to the framework regions of the original non-human antibody are selected as templates for CDR grafting.
  • a problem associated with antibody humanization by this method is the loss of affinity to their specific targets.
  • the simple grafting of CDR loops from murine antibodies onto human frameworks does not affect the antibody affinity, but in many cases it significantly reduces the binding affinity.
  • Some murine residues in framework regions referred to as vernier zone residues, have been demonstrated to affect the conformation of CDR loops and affinity of antibody (3). These residues are typically located in the P-sheet framework regions closely underlying the CDRs. Therefore, after the selection of desired human frameworks these residues are retained in the humanized antibody.
  • the Structural Annotation Index was developed, for example, to flag residues deemed important for maintaining the conformation and orientation of the CDRs (critical for antigen binding).
  • the SAI can provide a convenient and efficient alternative to the often tedious and time-consuming task of model building, structure inspection and mutational analysis, especially when there is a large number of antibodies to humanize or murinize.
  • variable domains (Fv) of immunoglobulins consists of 9 antiparallel -strands arranged into two sheets linked by a disulfide bond. These -strands, labelled A, B, C, C' , C" , D, E, F and G starting from the N-terminus and ending at the C- terminus, form a beta-sandwich known as the Ig-fold.
  • a two-dimensional topological diagram showing the connections between the P strands is shown in Fig. 2.
  • the 3 loops connecting the B and C strands (B-C loop), C' and C' strands (C' -C" loop), and F and G strands (F-G loop) define the hypervariable regions CDR1, CDR2 and CDR3 respectively, which typically form the antigen-binding sites on the antibody, or paratope.
  • four loops namely A-B, C-C' , C" -D and E-F lie on the end opposite the antigen-binding region and are therefore unlikely to directly affect antigen binding.
  • the fifth loop D-E loop which lies near CDR1 and/or CDR2, can influence antigen binding.
  • the SAI for residues in the query sequence can be automatically assigned by copying over the SAI at the corresponding Kabat positions in the aligned reference sequence. This vital point, which greatly facilitates the selection of back mutations, is discussed below.
  • Avastin® (Bevacizumab), a therapeutic biologic drug used in the treatment of various cancers, was used as a reference template for assigning SAI for other antibodies (described in Materials and Methods, Example 1).
  • SAI for the amino acids of Avastin® deduced from the X-ray structure are shown in Table 2 alongside the Kabat numbers.
  • Humira® (Adalimumab) whose X-ray structure is available, is used as an example.
  • Table 3 shows the Kabat number assignments for the residues in the heavy and light chains.
  • the SAI assignment for each residue in Humira® is made using the SAI corresponding to that Kabat number in Avastin (from Table 2), which is taken as the reference.
  • the thusly assigned SAI for Humira® was confirmed for correctness using the X-ray structure as a reference.
  • Humira® such assignments of SAI were verified for a number of therapeutic antibodies whose structures were available, thus establishing the legitimacy of the method.
  • a multiple sequence alignment of Avastin® with a number of clinical antibodies is shown in Table 4(a) and Table 4(b), which can use used for assigning SAI in a manner similar to Humira®.
  • Target-X xx mAbn Supk the various supernatants from the hybridomas.
  • X refers to the target (e.g., Target B)
  • xx is either “hz”, “hy” or “ch” depending on whether the construct is humanized (“hz”) in both the heavy and light chains, or a hybrid (“hy”) consisting of either a humanized heavy chain or a humanized light chain, or one in which neither the heavy nor the light chain is humanized (“ch”) and this has the same sequence as the chimera except for the mutations as noted
  • the “n” appearing in “mAbn” refers to the nth antibody against Target-X.
  • n ranges from 1 to 7
  • k in “Supk” refers to the kth construct in a given series.
  • Target-B_hz_mAb2_SuplO refers to the second series (mAb2) humanized (hz) construct 10 (Sup 10), for which data are shown in Figure 5B.
  • this particular construct has 5 back mutations in the heavy chain (R66K, V67A, M69L, T71 V, T73K) and a binding affinity for human Target-B (KD) of 4.5 nM.
  • Target-B Mice immunized with Target-B generated several antibodies, seven of which exhibited sub-micromolar binding affinities towards Target-B as assessed by SPR measurements.
  • the variable domains VH and VL of each one of these seven mAbs were compared with human germline sequences to find the optimal human homologs for humanization.
  • Tables 5 A through 5G The antibodies are named Target-B mAbl ch through Target-B_mAb7_ch and the designed clones follow the nomenclature described above.
  • Target B Antibodies mAbl, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7
  • Target-B_Hz_mAb2_Sup3 showed binding affinity of 4.8 nM which is comparable to the chimera Target-B_mAb2_Chimera (3.6 nM). For these reasons, it was the humanized construct of choice for the anti-Target B antibody Target-B_mAb2.
  • Target_B_Hz_mAb3_Sup3 showed more than a 5-fold loss in affinity (340 nM) compared to the murine chimera (63 nM).
  • framework mutations were selected based on the Structural Annotation Index were made to explore whether such variants could improve binding. In this case, conclusions based on the hybrid constructs carried over to the humanized constructs. On comparing Sup28 and Sup29, a 9-fold loss in affinity was noted in dropping the mutation A93S.
  • the humanized construct Sup30, with just two mutations in the heavy chain (M69L, A93V) showed a binding affinity of 50 nM, comparable to the chimera (63 nM) and was therefore prioritized.
  • the two humanized constructs corresponding to the two versions of the heavy chain with the same humanized light chain are denoted by Sup4 and Sup5. Looking at Sup2 and Sup5 which have the second version of the humanized heavy chain (the first version being Supl), it was observed that regardless of the light chain there was loss of binding. Given that the hybrid construct Sup3, which has the murine heavy chain but humanized light chain, showed binding (84 nM) comparable to the chimera, Target-B_mAb5_Chimera (86 nM), further investigations were made of humanized constructs with mutations in the heavy and light chains to determine whether these would conserve binding.
  • hybrids involving the humanized light chain (Sup2, Sup 10, and Sup 12) maintained chimera activity.
  • humanized constructs involving humanization of both the heavy and light chains did not show binding affinities comparable to the chimera unless several positions in the humanized heavy chain were back mutated to the murine sequence (e.g., Supl7, Supl8, Sup23, Sup24, Sup29, Sup30).
  • (vii) mAb7 The results for this set are shown in Figure 5G.
  • the binding affinity of the murine chimera is moderate to weak at 870 nM.
  • Hybrid versions with either the human chain humanized (Supl, Sup4, Sup5, Sup6) or the light chain humanized (Sup2, Sup7, Sup8, Sup9) were constructed, as well as the humanized version of both chains (Sup3).
  • the results for the hybrids show that humanized versions of the heavy chain showed binding affinities comparable to the chimera (Sup5, Sup6), whereas humanized versions of the light chain alone showed slightly better binding affinity towards Target-B (Sup7, Sup8, Sup9).
  • the SAI was developed to assess the extent to which framework residues in the variable heavy and light chains of monoclonal antibodies could be critical for maintaining the conformation of the CDRs and the tilt angle between the heavy and light chains.
  • a spreadsheet platform e.g., Microsoft Excel® or similar
  • the platform can be an interactive tool for humanization. It can facilitate mAb humanization by circumventing the need for building structural models for selecting residues for back mutagenesis.
  • the platform was used for the humanization of antibodies against protein Target-B, the latter leading to a biologic therapeutic drug. Guided by the SAI and the proposed VH/VL pairing suggestions, constructs were made that maintained the binding affinity and efficacy of the parent antibodies.
  • Figures 5 A-5G show binding data for murine antibodies and humanized constructs of anti-Target B antibodies. For further details, please see legend accompanying Table 3.
  • a database was prepared of human VH-VL pairings of human germlines using the VH and VL sequences of human and murine antibodies taken from the protein data bank (PDB, or www.rcsb.org) and clinical stage antibodies (Tushar Jain, Tingwan Sun, Stephanie Durandc, Amy Hall, Nga Rewa Houston, Juergen H. Nett, Beth Sharkey, Beata Bobrowicz, Isabelle Caffry, Yao Yu, Yuan Cao, Heather Lynaugh, Michael Brown, Hemanta Baruah, Laura T. Gray, Eric M. Krauland, Yingda Xu, Maximiliano Vasquez, and K. Dane Wittrup. Biophysical properties of the clinical-stage antibody landscape.
  • the mapped human germline VH and VL pairs thusly developed provided useful information towards designing antibody constructs that were more likely to express than constructs designed with no consideration given to the preferred tendencies of human germlines to pair.
  • antibodies designed with preferred VH-VL pairings showed better expression levels than those designed with little or no consideration to such pairings.

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Abstract

La présente demande concerne des procédés de conception et de modification de régions de structure pour des protéines d'anticorps, y compris des procédés de conception de séquences d'anticorps humanisés.
PCT/US2023/069480 2022-07-01 2023-06-30 Procédés d'humanisation d'anticorps WO2024006975A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006076288A2 (fr) 2005-01-11 2006-07-20 Five Prime Therapeutics, Inc. Constructions d'adn pour l'expression a long terme d'adn nu en injection intravasculaire
US20060270045A1 (en) 2003-10-22 2006-11-30 Keck Graduate Institute Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy
WO2008144757A1 (fr) * 2007-05-21 2008-11-27 Alder Biopharmaceuticals, Inc. Nouveaux procédés d'humanisation d'anticorps de lapin et anticorps de lapin humanisés
WO2011080350A1 (fr) * 2010-01-04 2011-07-07 Argen-X B.V. Anticorps humanisés

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060270045A1 (en) 2003-10-22 2006-11-30 Keck Graduate Institute Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy
WO2006076288A2 (fr) 2005-01-11 2006-07-20 Five Prime Therapeutics, Inc. Constructions d'adn pour l'expression a long terme d'adn nu en injection intravasculaire
WO2008144757A1 (fr) * 2007-05-21 2008-11-27 Alder Biopharmaceuticals, Inc. Nouveaux procédés d'humanisation d'anticorps de lapin et anticorps de lapin humanisés
WO2011080350A1 (fr) * 2010-01-04 2011-07-07 Argen-X B.V. Anticorps humanisés

Non-Patent Citations (38)

* Cited by examiner, † Cited by third party
Title
AL-LAZIKANI BLESK AMCHOTHIA C, J MOL BIOL., vol. 273, no. 4, 7 November 1997 (1997-11-07), pages 927 - 48
ASANOIZUMI KUMAGAI, JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 283, no. 2, 11 January 2008 (2008-01-11), pages 1156 - 1166
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CLAIRE MARKSALISSA M. HUMMERMARK CHINCHARLOTTE M. DEANE, BIOINFORMATICS, 2021, pages 1 - 7
CLARKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628
DALL'ACQUA W F ET AL: "Antibody humanization by framework shuffling", METHODS, ACADEMIC PRESS, NL, vol. 36, no. 1, 1 May 2005 (2005-05-01), pages 43 - 60, XP004852552, ISSN: 1046-2023, DOI: 10.1016/J.YMETH.2005.01.005 *
DING ZHANG ET AL: "A Novel Antibody Humanization Method Based on Epitopes Scanning and Molecular Dynamics Simulation", PLOS ONE, vol. 8, no. 11, 21 November 2013 (2013-11-21), pages e80636, XP055167980, DOI: 10.1371/journal.pone.0080636 *
DONDELINGER MFILEE PSAUVAGE EQUINTING BMUYLDERMANS SGALLENI MVANDEVENNE MS: "Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition", FRONT. IMMUNOL., vol. 9, 2018, pages 2278
ENDO ET AL., BIOTECHNOL. ADV., vol. 21, 2003, pages 695 - 713
FLATMAN ET AL., J. CHROMATOGR. B, vol. 848, 2007, pages 79 - 87
FOOTE JWINTER G.: "Antibody framework residues affecting the conformation of the hypervariable loops", J MOLBIOL., vol. 224, 1992, pages 487 - 99, XP024011188, DOI: 10.1016/0022-2836(92)91010-M
G. JOHNSONT.T. WU, NUCL. ACIDS RES., vol. 29, 2001, pages 205 - 206
GIUDICELLI V.CHAUME D.LEFRANC M.-P.: "IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes", NUCLEIC ACIDS RES., vol. 33, 2005, pages D256 - D261, XP055408999, DOI: 10.1093/narigki010
GIUDICELLI, V. ET AL., NUCLEIC ACIDS RES., vol. 33, 2005, pages D256 - D261
HOLLIGERHUDSON, NATURE BIOTECHNOLOGY, vol. 23, 2005, pages 1126 - 1136
I. RETTER ET AL., NUCL. ACIDS RES., vol. 33, no. 1, 2005, pages D671 - D674
JAMES R. APGAR ET AL: "Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody", MABS, vol. 8, no. 7, 13 September 2016 (2016-09-13), US, pages 1302 - 1318, XP055416065, ISSN: 1942-0862, DOI: 10.1080/19420862.2016.1215786 *
KABAT EA, TE, WU T, BILOFSKY H.: "Tabulation Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain BP-Microglobulins", HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH, 1979
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH, 1991
KARL J.M. HANF ET AL: "Antibody humanization by redesign of complementarity-determining region residues proximate to the acceptor framework", METHODS, vol. 65, no. 1, 1 January 2014 (2014-01-01), NL, pages 68 - 76, XP055416041, ISSN: 1046-2023, DOI: 10.1016/j.ymeth.2013.06.024 *
KINDT ET AL.: "Kuby Immunology", 2007, W.H. FREEMAN AND CO., pages: 91
LAURE-HÉLÈNE OUISSE ET AL: "Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies", BMC BIOTECHNOLOGY, vol. 17, no. 1, 9 December 2017 (2017-12-09), XP055548901, DOI: 10.1186/s12896-016-0322-5 *
LI B ET AL: "Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge", MABS, LANDES BIOSCIENCE, US, vol. 6, no. 5, 1 September 2014 (2014-09-01), pages 1255 - 1264, XP009185571, ISSN: 1942-0862, DOI: 10.4161/MABS.29809 *
M.P. LEFRANC, METHODS MOL. BIOL., vol. 248, 2004, pages 27 - 49
NARAYAN JAYARAMPALLAB BHOWMICKANDREW C.R.MARTIN: "Germline VH/VL pairing in antibodies", PROTEIN ENGINEERING, DESIGN & SELECTION, vol. 25, no. 10, 2012, pages 523 - 529
PORTOLANO ET AL., J. IMMUNOL., vol. 150, 1993, pages 880 - 887
RUNNING DEER ET AL., BIOTECHNOL. PROG., vol. 20, 2004, pages 880 - 889
SAFDARI YFARAJNIA SASGHARZADEH MKHALILI M.: "Antibody humanization methods - a review and update", BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS, vol. 9, no. 2, 2013, pages 175 - 186, XP055250530, DOI: 10.1080/02648725.2013.801235
SAMBROOK ET AL.: "Molecular Cloning, A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SITARAMAN ET AL., METHODS MOL. BIOL., vol. 498, 2009, pages 229 - 44
SPIRIN, RENDS BIOTECHNOL., vol. 22, 2004, pages 538 - 45
THOMAS TILLERINGRID SCHUSTERDOROTHEE DEPPEKATJA SIEGERSRALF STROHNERTANJA HERRMANNMARION BERENGUERDOMINIQUE POUJOLJENNIFER STEHLEY: "A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties", MABS, vol. 5, no. 3, 2013, pages 445 - 470, XP055377037, DOI: 10.4161/mabs.24218
TOBIAS H. OLSENFERGUS BOYLESHARLOTTE M. DEANE: "Observed Antibody Space: A diverse database of cleaned, annotated, and translated unpaired and paired antibody sequences", PROTEIN SCIENCE, 2021, pages 1 - 6
TUSHAR JAINTINGWAN SUNSTEPHANIE DURANDCAMY HALLNGA REWA HOUSTONJUERGEN H. NETTBETH SHARKEYBEATA BOBROWICZISABELLE CAFFRYYAO YU: "Biophysical properties of the clinical-stage antibody landscape", PNAS, vol. 114, no. 5, 31 January 2017 (2017-01-31), pages 944 - 949
VAHIDEH AHMADZADEH ET AL: "Antibody Humanization Methods for Development of Therapeutic Applications", MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY, vol. 33, no. 2, 1 April 2014 (2014-04-01), US, pages 67 - 73, XP055385027, ISSN: 2167-9436, DOI: 10.1089/mab.2013.0080 *
YAGHOUB SAFDARI ET AL: "Antibody humanization methods : a review and update", BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS, vol. 29, no. 2, 1 October 2013 (2013-10-01), GB, pages 175 - 186, XP055250530, ISSN: 0264-8725, DOI: 10.1080/02648725.2013.801235 *
YOONJOO CHOICASEY HUACHARLES L SENTMANMARGARET E ACKERMANCHRIS BAILEY-KELLOGG: "Antibody humanization by structure-based computational protein design", MABS, vol. 7, no. 6, 2015, pages 1045 - 1057, XP055250542, DOI: 10.1080/19420862.2015.1076600

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