US20140170152A1 - Immunobinders directed against tnf - Google Patents

Immunobinders directed against tnf Download PDF

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US20140170152A1
US20140170152A1 US14/073,479 US201314073479A US2014170152A1 US 20140170152 A1 US20140170152 A1 US 20140170152A1 US 201314073479 A US201314073479 A US 201314073479A US 2014170152 A1 US2014170152 A1 US 2014170152A1
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seq
disorder
tnf
residues
cdr
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US14/073,479
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Chung-Ming Hsieh
Lorenzo Benatuil
Yuliya Kutskova
John Memmott
Jennifer Perez
Suju Zhong
Carrie Goodreau
Anca Clabbers
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AbbVie Inc
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AbbVie Inc
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Priority to US14/073,479 priority Critical patent/US20140170152A1/en
Publication of US20140170152A1 publication Critical patent/US20140170152A1/en
Assigned to ABBVIE INC. reassignment ABBVIE INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GOODREAU, CARRIE, MEMMOTT, John, ZHONG, SUJU, BENATUIL, LORENZO, CLABBERS, ANCA, HSIEH, CHUNG-MING, KUTSKOVA, YULIYA, PEREZ, JENNIFER
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    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/464—Igs containing CDR-residues from one specie grafted between FR-residues from another
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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Definitions

  • TNF- ⁇ binding proteins and their uses in the prevention and/or treatment of acute and chronic immunological diseases are provided.
  • TNF- ⁇ also referred to as tumor necrosis factor, tumor necrosis factor-alpha, tumor necrosis factor- ⁇ , TNF, and cachectin.
  • TNF- ⁇ also referred to as tumor necrosis factor, tumor necrosis factor-alpha, tumor necrosis factor- ⁇ , TNF, and cachectin.
  • the antigen binding domain comprises the VH region chosen from any one of SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34-58, 74-83, 94-266, 478-486, 496-675, 738-762, 778-956, 1053-1062, 1073, 1075, and 1077, or one, two, or three CDRs therefrom.
  • the antigen binding domain comprises the VL region chosen from any one of SEQ ID NOs: 23, 25, 27, 29, 31, 33, 59-73, 84-93, 267-477, 487-495, 676-737, 763-777, 957-1052, 1063-1072, 1074, 1076, and 1078, or one, two, or three CDRs therefrom.
  • the antigen binding domain comprises a VH region and a VL region, for example, wherein the VH region comprises SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34-58, 74-83, 94-266, 478-486, 496-675, 738-762, 778-956, 1053-1062, 1073, 1075, and 1077, or one, two, or three CDRs therefrom, and the VL region comprises SEQ ID NOs: 23, 25, 27, 29, 31, 33, 59-73, 84-93, 267-477, 487-495, 676-737, 763-777, 957-1052, 1063-1072, 1074, 1076, and 1078, or one, two, or three CDRs therefrom.
  • the VH region comprises SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34-58, 74-83, 94-266, 478-486, 496-675, 738-762, 778-956, 1053-1062, 1073, 1075, and 1077,
  • the binding protein binds TNF- ⁇ . In another embodiment, the binding protein modulates a biological function of TNF- ⁇ . In another embodiment, the binding protein neutralizes TNF- ⁇ . In yet another embodiment, the binding protein diminishes the ability of TNF- ⁇ to bind to its receptor, for example, the binding protein diminishes the ability of pro-human TNF- ⁇ , mature-human TNF- ⁇ , or truncated-human TNF- ⁇ to bind to its receptor. In yet another embodiment, the binding protein reduces one or more TNF- ⁇ biological activities selected from: TNF-dependent cytokine production; TNF-dependent cell killing; TNF-dependent inflammation; TNF-dependent bone erosion; and TNF-dependent cartilage damage.
  • the binding protein has an on rate constant (K on ) selected from: at least about 10 2 M ⁇ 1 s ⁇ 1 ; at least about 10 3 M ⁇ 1 s ⁇ 1 ; at least about 10 4 M ⁇ 1 s ⁇ 1 ; at least about 10 5 M ⁇ 1 s ⁇ 1 ; and at least about 10 6 M ⁇ 1 s ⁇ 1 ; as measured by surface plasmon resonance.
  • K on on rate constant
  • the binding protein has an off rate constant (K off ) selected from: at most about 10 ⁇ 3 s ⁇ 1 ; at most about 10 ⁇ 4 s ⁇ 1 ; at most about 10 ⁇ 5 s ⁇ 1 ; and at most about 10 ⁇ 6 s ⁇ 1 , as measured by surface plasmon resonance.
  • the binding protein has a dissociation constant (K D ) selected from: at most about 10 ⁇ 7 M; at most about 10 ⁇ 8 M; at most about 10 ⁇ 9 M; at most about 10 ⁇ 10 M; m at most about 10 ⁇ 11 M; at most about 10 ⁇ 12 M; and at most 10 ⁇ 13 M.
  • a method for treating a mammal comprising administering to the mammal an effective amount of the pharmaceutical composition disclosed herein.
  • a method for reducing human TNF- ⁇ activity is provided, the method comprising: contacting human TNF- ⁇ with the binding protein disclosed herein such that human TNF- ⁇ activity is reduced.
  • a method for reducing human TNF- ⁇ activity in a human subject suffering from a disorder in which TNF- ⁇ activity is detrimental the method comprising administering to the human subject the binding protein disclosed herein such that human TNF- ⁇ activity in the human subject is reduced.
  • a method for treating a subject for a disease or a disorder in which TNF- ⁇ activity is detrimental the method comprising administering to the subject the binding protein disclosed herein such that treatment is achieved.
  • the method treats diseases involving immune and inflammatory elements, such as autoimmune diseases, particularly those associated with inflammation, including Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoriatic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, and ankylosing spondylitis. Therefore, the binding proteins herein may be used to treat these disorders.
  • autoimmune diseases particularly those associated with inflammation, including Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoriatic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, and ankylosing spondylitis. Therefore, the binding proteins herein may be used to treat these disorders.
  • TNF- ⁇ binding proteins or antigen-binding portions thereof, that bind TNF- ⁇ , pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such binding proteins and fragments. Also provided are methods of using the binding proteins disclosed herein to detect human TNF- ⁇ , to inhibit human TNF- ⁇ either in vitro or in vivo, and to regulate gene expression or TNF- ⁇ related functions.
  • human TNF- ⁇ (abbreviated herein as hTNF- ⁇ ) includes a trimeric cytokine protein.
  • the term includes a homotrimeric protein comprising three 17.5 kD TNF- ⁇ proteins.
  • the homotrimeric protein is referred to as a “TNF- ⁇ protein”.
  • human “TNF- ⁇ ” is intended to include recombinant human TNF- ⁇ (rhTNF- ⁇ ), which can be prepared by standard recombinant expression methods.
  • the sequence of human TNF- ⁇ is shown in Table 1.
  • antibody broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule.
  • Ig immunoglobulin
  • L light
  • mutant, variant, or derivative antibody formats are known in the art.
  • each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.
  • binding protein portion refers to one or more fragments of a binding protein that retain the ability to specifically bind to an antigen (e.g., hTNF- ⁇ ).
  • the antigen-binding function of a binding protein can be performed by fragments of a full-length binding protein.
  • binding protein embodiments may also have bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens.
  • binding fragments encompassed within the term “antigen-binding portion” of a binding protein include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.
  • VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci.
  • scFv single chain Fv
  • Single chain binding proteins are also intended to be encompassed within the term “antigen-binding portion” of a binding protein.
  • Other forms of single chain binding proteins, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific binding proteins in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, e.g., Holliger, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, et al. (1994) Structure 2:1121-1123).
  • binding protein refers to a polypeptide comprising one or more antigen-binding portions disclosed herein optionally linked to a linker polypeptide or a constant domain.
  • Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions.
  • linker polypeptides are well known in the art (see e.g., Holliger, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, et al. (1994) Structure 2:1121-1123).
  • a constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art and represented in Table 2.
  • a binding protein, or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the binding protein or binding protein portion with one or more other proteins or peptides.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, et al. (1995) Hum. Antibod. Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, et al. (1994) Mol. Immunol.
  • Antibody portions such as Fab and F(ab′) 2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies.
  • binding proteins, binding protein portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
  • an “isolated binding protein” refers to a binding protein, or antigen-binding portion thereof, that is substantially free of other binding proteins having different antigenic specificities (e.g., an isolated binding protein that specifically binds hTNF- ⁇ is substantially free of binding proteins that specifically bind antigens other than hTNF- ⁇ ).
  • An isolated binding protein that specifically binds hTNF- ⁇ may, however, have cross-reactivity to other antigens, such as TNF- ⁇ molecules from other species.
  • an isolated binding protein may be substantially free of other cellular material and/or chemicals.
  • human binding protein includes binding proteins, or antigen-binding portion thereof, that having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human binding proteins disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term “human binding protein” is not intended to include binding proteins in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • Kabat numbering “Kabat definitions” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 106 for CDR3.
  • the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
  • CDR refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions.
  • CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.
  • CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.
  • the methods used herein may utilize CDRs defined according to any of these systems, although particular embodiments use Kabat or Chothia defined CDRs.
  • Human heavy chain and light chain acceptor sequences are known in the art.
  • the human heavy chain and light chain acceptor sequences are selected from the sequences listed from V-base (hvbase.mrc-cpe.cam.ac.uk/) or from IMGT®, the international ImMunoGeneTics information system® (himgt.cines.fr/textes/IMGTrepertoire/LocusGenes/).
  • the human heavy chain and light chain acceptor sequences are selected from the sequences described in Table 3 and Table 4, respectively.
  • multivalent binding protein is used in this specification to denote a binding protein comprising two or more antigen binding sites.
  • the multivalent binding protein may be engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody.
  • multispecific binding protein refers to a binding protein capable of binding two or more related or unrelated targets.
  • Dual variable domain (DVD) binding proteins or immunoglobulins (DVD-Ig) as used herein are binding proteins that comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins.
  • DVD-binding proteins may be monospecific, i.e., capable of binding one antigen or multispecific, i.e., capable of binding two or more antigens.
  • DVD-binding proteins comprising two heavy chain DVD-Ig polypeptides and two light chain DVD-Ig polypeptides are referred to a DVD-Ig.
  • Each half of a DVD-Ig comprises a heavy chain DVD-Ig polypeptide, and a light chain DVD-Ig polypeptide, and two antigen binding sites.
  • Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site.
  • DVD binding proteins and methods of making DVD binding proteins are disclosed in U.S. Pat. No. 7,612,181.
  • DVD binding protein comprising binding proteins capable of binding TNF- ⁇ .
  • the DVD binding protein is capable of binding TNF- ⁇ and a second target.
  • neutralizing refers to neutralization of a biological activity of a cytokine when a binding protein specifically binds the cytokine.
  • binding of a neutralizing binding protein to hTNF- ⁇ results in inhibition of a biological activity of hTNF- ⁇ , e.g., the neutralizing binding protein binds hTNF- ⁇ and reduces a biologically activity of hTNF- ⁇ by at least about 20%, 40%, 60%, 80%, 85% or more
  • Inhibition of a biological activity of hTNF- ⁇ by a neutralizing binding protein can be assessed by measuring one or more indicators of hTNF- ⁇ biological activity well known in the art. For example neutralization of the cytoxicity of TNF- ⁇ on L929 cells.
  • the terms “agonist” or “agonizing” refer to an increase of a biological activity of TNF- ⁇ when a binding protein specifically binds TNF- ⁇ , e.g., hTNF- ⁇ .
  • binding of an agonizing binding protein to TNF- ⁇ results in the increase of a biological activity of TNF- ⁇ .
  • the agonistic binding protein binds TNF- ⁇ and increases a biologically activity of TNF- ⁇ by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95, 96%, 97%, 98%, 99%, and 100%.
  • An inhibition of a biological activity of TNF- ⁇ by an agonistic binding protein can be assessed by measuring one or more indicators of TNF- ⁇ biological activity well known in the art.
  • activity includes activities such as the binding specificity/affinity of a binding protein for an antigen, for example, a hTNF- ⁇ binding protein that binds to a TNF- ⁇ antigen and/or the neutralizing potency (or agonizing potency) of a binding protein, for example, a hTNF- ⁇ binding protein whose binding to hTNF- ⁇ inhibits the biological activity of hTNF- ⁇ , e.g., neutralization of the cytoxicity of TNF- ⁇ on L929 cells.
  • activities such as the binding specificity/affinity of a binding protein for an antigen, for example, a hTNF- ⁇ binding protein that binds to a TNF- ⁇ antigen and/or the neutralizing potency (or agonizing potency) of a binding protein, for example, a hTNF- ⁇ binding protein whose binding to hTNF- ⁇ inhibits the biological activity of hTNF- ⁇ , e.g., neutralization of the cytoxicity of TNF
  • surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).
  • K on refers to the on rate constant for association of a binding protein (e.g., an antibody) to the antigen to form, e.g., the antibody/antigen complex as is known in the art.
  • the “K on ” also is known by the terms “association rate constant”, or “ka”, as used interchangeably herein. This value indicating the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen also is shown by the equation below:
  • K off refers to the off rate constant for dissociation, or “dissociation rate constant”, of a binding protein (e.g., an antibody), from the, e.g., antibody/antigen complex as is known in the art. This value indicates the dissociation rate of an antibody from its target antigen or separation of Ab ⁇ Ag complex over time into free antibody and antigen as shown by the equation below:
  • K D refers to the “equilibrium dissociation constant” and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (K off ) by the association rate constant (K on ).
  • the association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium.
  • BIAcore® biological interaction analysis
  • KinExA® KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used.
  • One aspect of the present disclosure provides isolated fully-human anti-human TNF binding proteins, such as monoclonal antibodies, or antigen-binding portions thereof, that bind to TNF- ⁇ with high affinity, a slow off rate and high neutralizing capacity.
  • a second aspect of the disclosure provides affinity-matured fully-human anti-TNF binding proteins, such as monoclonal antibodies, or antigen-binding portions thereof, that bind to TNF- ⁇ with high affinity, a slow off rate and high neutralizing capacity.
  • binding proteins disclosed herein may be made by any of a number of techniques known in the art.
  • binding proteins are produced by immunizing a non-human animal comprising some, or all, of the human immunoglobulin locus with a TNF- ⁇ antigen.
  • the non-human animal is a XENOMOUSE transgenic mouse, an engineered mouse strain that comprises large fragments of the human immunoglobulin loci and is deficient in mouse antibody production. See, e.g., Green et al. (1994) Nature Genet. 7:13-21 and U.S. Pat. Nos. 5,916,771; 5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598 and 6,130,364.
  • the XENOMOUSE transgenic mouse contains approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and x light chain loci. See, Mendez et al. (1997) Nature Genet. 15:146-156; Green and Jakobovits (1998) J. Exp. Med. 188:483-495.
  • In vitro methods also can be used to make the binding protein disclosed herein, wherein an antibody library is screened to identify an antibody having the desired binding specificity.
  • Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, U.S. Pat. No. 5,223,409; PCT Publications WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 97/29131; Fuchs et al. (1991) Bio/Technology 9:1369-1372; Hay et al. (1992) Hum. Antibod.
  • the recombinant antibody library may be from a subject immunized with TNF- ⁇ , or a portion of TNF- ⁇ .
  • the recombinant antibody library may be from a na ⁇ ve subject, i.e., one who has not been immunized with TNF- ⁇ , such as a human antibody library from a human subject who has not been immunized with human TNF- ⁇ .
  • Antibodies disclosed herein are selected by screening the recombinant antibody library with the peptide comprising human TNF- ⁇ to thereby select those antibodies that recognize TNF- ⁇ . Methods for conducting such screening and selection are well known in the art, such as described in the references in the preceding paragraph.
  • an isolated binding protein or an antigen-binding portion thereof, that binds TNF- ⁇ , e.g., human TNF- ⁇ .
  • the binding protein is a neutralizing binding protein.
  • the binding protein is a recombinant binding protein or a monoclonal antibody.
  • the binding proteins disclosed herein can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the binding proteins disclosed herein can be found in the art.
  • the binding protein coding regions from the phage can be isolated and used to generate whole binding proteins including human binding protein or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • techniques to recombinantly produce Fab, Fab′ and F(ab′) 2 fragments can also be employed using methods known in the art such as those disclosed in PCT Publication WO 92/22324; Mullinax et al. (1992) BioTechniques 12(6):864-869; and Sawai et al. (1995) Am. J. Reprod. Immunol.
  • RNA-protein fusions as described in PCT Publication No. WO 98/31700 and in Roberts and Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302.
  • a covalent fusion is created between an mRNA and the peptide or protein that it encodes by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end.
  • a specific mRNA can be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein, e.g., antibody, or portion thereof, such as binding of the antibody, or portion thereof, to the dual specificity antigen.
  • mRNAs e.g., a combinatorial library
  • Nucleic acid sequences encoding antibodies, or portions thereof, recovered from screening of such libraries can be expressed by recombinant means as described above (e.g., in mammalian host cells) and, moreover, can be subjected to further affinity maturation by either additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence(s), or by other methods for affinity maturation in vitro of recombinant antibodies, as described above.
  • the binding proteins disclosed herein can also be generated using yeast display methods known in the art.
  • yeast display methods genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of yeast.
  • yeast can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • yeast display methods that can be used to make the binding proteins disclosed herein include those disclosed Wittrup et al. U.S. Pat. No. 6,699,658 and Frenken et al., U.S. Pat. No. 6,114,147.
  • Binding proteins disclosed herein may be produced by any of a number of techniques known in the art. For example, expression from host cells, wherein expression vector(s) encoding the heavy and light chains is (are) transfected into a host cell by standard techniques.
  • the various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • binding protein in either prokaryotic or eukaryotic host cells
  • expression of binding protein in eukaryotic cells is contemplated, for example, in mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active binding protein.
  • Mammalian host cells for expressing the recombinant binding proteins disclosed herein include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) J. Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2 cells.
  • Chinese Hamster Ovary CHO cells
  • dhfr-CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) J. Mol. Biol. 159:601-621
  • NS0 myeloma cells COS cells and SP2 cells.
  • binding proteins When recombinant expression vectors encoding binding protein genes are introduced into mammalian host cells, the binding proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the binding protein in the host cells or, in particular, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
  • Host cells can also be used to produce functional binding protein fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and/or the heavy chain of a binding protein disclosed herein. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the binding proteins disclosed herein.
  • bifunctional binding proteins may be produced in which one heavy and one light chain are a binding protein disclosed herein and the other heavy and light chain are specific for an antigen other than the antigens of interest by crosslinking a binding protein disclosed herein to a second binding protein by standard chemical crosslinking methods.
  • a recombinant expression vector encoding both the heavy chain and the light chain is introduced into dhfr ⁇ CHO cells by calcium phosphate-mediated transfection.
  • the heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes.
  • the recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification.
  • the selected transformant host cells are cultured to allow for expression of the heavy and light chains and intact binding protein is recovered from the culture medium.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the binding protein from the culture medium.
  • a method of synthesizing a recombinant binding protein disclosed herein is provided by culturing a host cell disclosed herein in a suitable culture medium until a recombinant binding protein disclosed herein is synthesized. The method can further comprise isolating the recombinant binding protein from the culture medium.
  • Table 5 provides the VH and VL sequences of fully human anti-human TNF binding proteins, including CDRs from each VH and VL sequence.
  • Table 6 provides the VH sequence of humanized anti-TNF MAK-195 antibodies that were converted into IgG clones as discussed in detail in Example 2.
  • Table 7 provides VL sequences of IgG converted clones for Humanized anti-TNF MAK-195 antibodies as discussed in detail in Example 2.
  • Table 8 provides VH sequences of humanized anti-TNF MAK-199 converted clones as discussed in detail in Example 3.
  • Table 9 provides VL sequences of humanized anti-TNF MAK-199 converted clones as discussed in detail in Example 3.
  • the antigen binding domain comprises the VH region chosen from any one of SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34-58, 74-83, 94-266, 478-486, 496-675, 738-762, 778-956, 1053-1062, 1073, 1075, and 1077, or one, two, or three CDRs therefrom.
  • the antigen binding domain comprises the VL region chosen from any one of SEQ ID NOs: 23, 25, 27, 29, 31, 33, 59-73, 84-93, 267-477, 487-495, 676-737, 763-777, 957-1052, 1063-1072, 1074, 1076, and 1078, or one, two, or three CDRs therefrom.
  • the antigen binding domain comprises a VH region and a VL region, for example, wherein the VH region comprises SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34-58, 74-83, 94-266, 478-486, 496-675, 738-762, 778-956, 1053-1062, 1073, 1075, and 1077, or one, two, or three CDRs therefrom, and the VL region comprises SEQ ID NOs: 23, 25, 27, 29, 31, 33, 59-73, 84-93, 267-477, 487-495, 676-737, 763-777, 957-1052, 1063-1072, 1074, 1076, and 1078, or one, two, or three CDRs therefrom.
  • the VH region comprises SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34-58, 74-83, 94-266, 478-486, 496-675, 738-762, 778-956, 1053-1062, 1073, 1075, and 1077,
  • the human acceptor framework comprises at least one amino acid sequence selected from: SEQ ID NOs: 6-21.
  • the human acceptor framework comprises an amino acid sequence selected from: SEQ 1N NOs: 9, 10, 11, 12, 15, 16, 17, and 21.
  • the human acceptor framework comprises at least one framework region amino acid substitution, wherein the amino acid sequence of the framework is at least 65% identical to the sequence of the human acceptor framework and comprises at least 70 amino acid residues identical to the human acceptor framework.
  • the human acceptor framework comprises at least one framework region amino acid substitution at a key residue.
  • the key residue selected from: a residue adjacent to a CDR; a glycosylation site residue; a rare residue; a residue capable of interacting with human TNF- ⁇ ; a residue capable of interacting with a CDR; a canonical residue; a contact residue between heavy chain variable region and light chain variable region; a residue within a Vernier zone; and a residue in a region that overlaps between a Chothia-defined variable heavy chain CDR1 and a Kabat-defined first heavy chain framework.
  • the key residue is selected from: H1, H12, H24, H27, H29, H37, H48, H49, H67, H71, H73, H76, H78, L13, L43, L58, L70, and L80.
  • the VH mutation is selected from: Q1E, I12V, A24V, G27F, I29L, V29F F29L 137V, I48L, V48L, S49G, V67L, F67L, V71K, R71K, T73N, N76S, L78I, and F78I.
  • the VL mutation is selected from: V13L, A435, I58V, E70D, and S80P.
  • the binding protein comprises two variable domains, wherein the two variable domains have amino acid sequences selected from: SEQ ID NOS: 22 and 23; 23 and 24; 24 and 25; 26 and 27; 28 and 29; 30 and 31; or 32 and 33.
  • TNF- ⁇ binding proteins disclosed herein exhibit a high capacity to reduce or to neutralize TNF- ⁇ activity, e.g., as assessed by any one of several in vitro and in vivo assays known in the art.
  • TNF- ⁇ binding proteins disclosed herein also exhibit a high capacity to increase or agonize TNF- ⁇ activity.
  • the isolated binding protein, or antigen-binding portion thereof binds human TNF- ⁇ , wherein the binding protein, or antigen-binding portion thereof, dissociates from human TNF- ⁇ with a k off rate constant of about 0.1 s ⁇ 1 or less, as determined by surface plasmon resonance, such as 1 ⁇ 10 ⁇ 2 s ⁇ 1 or less, 1 ⁇ 10 ⁇ 3 s ⁇ 1 or less, 1 ⁇ 10 ⁇ 4 s ⁇ 1 or less, 1 ⁇ 10 ⁇ 5 s ⁇ 1 or less and 1 ⁇ 10 ⁇ 6 s ⁇ 1 or less; or which inhibits human TNF- ⁇ activity with an IC 50 of about 1 ⁇ 10 ⁇ 6 M or less, such as 1 ⁇ 10 ⁇ 7 M or less, 1 ⁇ 10 ⁇ 8 M or less, 1 ⁇ 10 ⁇ 9 M or less, 1 ⁇ 10 ⁇ 10 M or less and 1 ⁇ 10 11 M or less.
  • a k off rate constant of about 0.1 s ⁇ 1 or less, as determined by surface plasmon resonance, such
  • the binding protein comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region.
  • the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region.
  • the binding protein can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region.
  • the binding protein comprises a kappa light chain constant region.
  • the binding protein portion can be, for example, a Fab fragment or a single chain Fv fragment.
  • the Fc portion of a binding protein mediates several important effector functions, e.g., cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives.
  • Neonatal Fc receptors are the critical components determining the circulating half-life of antibodies.
  • at least one amino acid residue is replaced in the constant region of the binding protein, for example the Fc region of the binding protein, such that effector functions of the binding protein are altered.
  • a labeled binding protein wherein an antibody or antibody portion disclosed herein is derivatized or linked to another functional molecule (e.g., another peptide or protein).
  • a labeled binding protein disclosed herein can be derived by functionally linking an antibody or antibody portion disclosed herein (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • Useful detectable agents with which an antibody or antibody portion disclosed herein may be derivatized include fluorescent compounds.
  • Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like.
  • An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product.
  • the detectable agent horseradish peroxidase when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable.
  • An antibody may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
  • Another embodiment of the disclosure provides a crystallized binding protein.
  • the crystallized binding protein has a greater half-life in vivo than the soluble counterpart of the binding protein.
  • the binding protein retains biological activity after crystallization.
  • Crystallized binding protein disclosed herein may be produced according methods known in the art and as disclosed in PCT Publication WO 02/72636.
  • Another embodiment of the disclosure provides a glycosylated binding protein wherein the binding protein or antigen-binding portion thereof comprises one or more carbohydrate residues.
  • Nascent in vivo protein production may undergo further processing, known as post-translational modification.
  • sugar (glycosyl) residues may be added enzymatically, a process known as glycosylation.
  • glycosylation The resulting proteins bearing covalently linked oligosaccharide side chains are known as glycosylated proteins or glycoproteins. Protein glycosylation depends on the amino acid sequence of the protein of interest, as well as the host cell in which the protein is expressed.
  • glycosylation enzymes e.g., glycosyltransferases and glycosidases
  • substrates nucleotide sugars
  • protein glycosylation pattern, and composition of glycosyl residues may differ depending on the host system in which the particular protein is expressed.
  • Glycosyl residues useful in the disclosure may include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid.
  • the glycosylated binding protein comprises glycosyl residues such that the glycosylation pattern is human.
  • a therapeutic protein produced in a microorganism host such as yeast
  • glycosylated utilizing the yeast endogenous pathway may be reduced compared to that of the same protein expressed in a mammalian cell, such as a CHO cell line.
  • Such glycoproteins may also be immunogenic in humans and show reduced half-life in vivo after administration.
  • Specific receptors in humans and other animals may recognize specific glycosyl residues and promote the rapid clearance of the protein from the bloodstream.
  • a practitioner may prefer a therapeutic protein with a specific composition and pattern of glycosylation, for example glycosylation composition and pattern identical, or at least similar, to that produced in human cells or in the species-specific cells of the intended subject animal.
  • Expressing glycosylated proteins different from that of a host cell may be achieved by genetically modifying the host cell to express heterologous glycosylation enzymes. Using techniques known in the art a practitioner may generate antibodies or antigen-binding portions thereof exhibiting human protein glycosylation. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylation enzymes such that glycosylated proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation identical to that of animal cells, especially human cells (U.S. Pat. Nos. 7,449,308 and 7,029,872).
  • a protein of interest may be expressed using a library of host cells genetically engineered to express various glycosylation enzymes, such that member host cells of the library produce the protein of interest with variant glycosylation patterns. A practitioner may then select and isolate the protein of interest with particular novel glycosylation patterns. In an embodiment, the protein having a particularly selected novel glycosylation pattern exhibits improved or altered biological properties.
  • the human TNF- ⁇ binding proteins, or portions thereof, disclosed herein can be used to detect TNF- ⁇ (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry.
  • a conventional immunoassay such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry.
  • ELISA enzyme linked immunosorbent assays
  • RIA radioimmunoassay
  • a method for detecting TNF- ⁇ in a biological sample comprising contacting a biological sample with a binding protein, or binding protein portion, disclosed herein and detecting either the binding protein (or binding protein portion) bound to TNF- ⁇ or unbound binding protein (or binding protein portion), to thereby detect TNF- ⁇ in the biological sample.
  • the binding protein is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody.
  • Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • suitable radioactive material include 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153
  • human TNF- ⁇ can be assayed in biological fluids by a competition immunoassay utilizing rhTNF- ⁇ standards labeled with a detectable substance and an unlabeled human TNF- ⁇ binding protein.
  • the biological sample, the labeled rhTNF- ⁇ standards and the human TNF- ⁇ binding protein are combined and the amount of labeled rhTNF- ⁇ standard bound to the unlabeled binding protein is determined.
  • the amount of human TNF- ⁇ in the biological sample is inversely proportional to the amount of labeled rhTNF- ⁇ standard bound to the TNF- ⁇ binding protein.
  • human TNF- ⁇ can also be assayed in biological fluids by a competition immunoassay utilizing rhTNF- ⁇ standards labeled with a detectable substance and an unlabeled human TNF- ⁇ binding protein.
  • the binding proteins and binding protein portions disclosed herein are capable of neutralizing TNF- ⁇ activity, e.g., human TNF- ⁇ activity, both in vitro and in vivo.
  • the binding proteins and binding protein portions disclosed herein are capable of increasing or agonizing human TNF- ⁇ activity, e.g., human TNF- ⁇ activity. Accordingly, such binding proteins and binding protein portions disclosed herein can be used to inhibit or increase hTNF- ⁇ activity, e.g., in a cell culture containing hTNF- ⁇ , in human subjects or in other mammalian subjects having TNF- ⁇ with which a binding protein disclosed herein cross-reacts.
  • a method for inhibiting or increasing hTNF- ⁇ activity comprising contacting hTNF- ⁇ with a binding protein or binding protein portion disclosed herein such that hTNF- ⁇ activity is inhibited or increased.
  • a binding protein or binding protein portion disclosed herein can be added to the culture medium to inhibit or increase hTNF- ⁇ activity in the culture.
  • a method for reducing or increasing hTNF- ⁇ activity in a subject advantageously from a subject suffering from a disease or disorder in which TNF- ⁇ activity is detrimental or, alternatively, beneficial.
  • Methods for reducing or increasing TNF- ⁇ activity in a subject suffering from such a disease or disorder comprises administering to the subject a binding protein or binding protein portion disclosed herein such that TNF- ⁇ activity in the subject is reduced or increased.
  • the TNF- ⁇ is human TNF- ⁇
  • the subject is a human subject.
  • the subject can be a mammal expressing a TNF- ⁇ to which a binding protein provided is capable of binding.
  • the subject can be a mammal into which TNF- ⁇ has been introduced (e.g., by administration of TNF- ⁇ or by expression of a TNF- ⁇ transgene).
  • a binding protein disclosed herein can be administered to a human subject for therapeutic purposes.
  • a binding protein disclosed herein can be administered to a non-human mammal expressing a TNF- ⁇ with which the binding protein is capable of binding for veterinary purposes or as an animal model of human disease.
  • animal models may be useful for evaluating the therapeutic efficacy of binding proteins disclosed herein (e.g., testing of dosages and time courses of administration).
  • a disorder in which TNF- ⁇ activity is detrimental includes diseases and other disorders in which the presence of TNF- ⁇ activity in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which TNF- ⁇ activity is detrimental is a disorder in which reduction of TNF- ⁇ activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of TNF- ⁇ in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of TNF- ⁇ in serum, plasma, synovial fluid, etc. of the subject), which can be detected, for example, using an anti-TNF- ⁇ antibody as described above.
  • disorders that can be treated with the binding proteins disclosed herein include those disorders discussed in the section below pertaining to pharmaceutical compositions of the antibodies disclosed herein.
  • a disorder in which TNF- ⁇ activity is beneficial include diseases and other disorders in which the presence of TNF- ⁇ activity in a subject suffering from the disorder has been shown to be or is suspected of being either beneficial for treating the pathophysiology of the disorder or a factor that contributes to a treatment of the disorder. Accordingly, a disorder in which TNF- ⁇ activity is beneficial is a disorder in which an increase of TNF- ⁇ activity is expected to alleviate the symptoms and/or progression of the disorder.
  • disorders that can be treated with the antibodies disclosed herein include those disorders discussed in the section below pertaining to pharmaceutical compositions of the antibodies disclosed herein.
  • compositions comprising a binding protein, or antigen-binding portion thereof, disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions comprising binding protein disclosed herein are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder, in preventing, treating, managing, or ameliorating of a disorder or one or more symptoms thereof, and/or in research.
  • a composition comprises one or more binding proteins disclosed herein.
  • the pharmaceutical composition comprises one or more binding proteins disclosed herein and one or more prophylactic or therapeutic agents other than binding proteins disclosed herein for treating a disorder in which TNF- ⁇ activity is detrimental.
  • the composition may further comprise of a carrier, diluent or excipient.
  • the binding proteins and binding protein-portions disclosed herein can be incorporated into pharmaceutical compositions suitable for administration to a subject.
  • the pharmaceutical composition comprises a binding protein or binding protein portion disclosed herein and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition, may be included.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding protein or binding protein portion.
  • Various delivery systems are known and can be used to administer one or more binding proteins disclosed herein or the combination of one or more binding proteins disclosed herein and a prophylactic agent or therapeutic agent useful for preventing, managing, treating, or ameliorating a disorder or one or more symptoms thereof, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the binding protein or binding protein fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu (1987) J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc.
  • a prophylactic agent or therapeutic agent useful for preventing, managing, treating, or ameliorating a disorder or one or more symptoms thereof, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the binding protein or binding protein fragment, receptor-mediated endocytos
  • Methods of administering a prophylactic or therapeutic agent disclosed herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes).
  • parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous
  • epidural administration e.g., intratumoral administration
  • mucosal administration e.g., intranasal and oral routes
  • pulmonary administration can be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • a binding protein disclosed herein, combination therapy, or a composition disclosed herein is administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).
  • prophylactic or therapeutic agents disclosed herein are administered intramuscularly, intravenously, intratumorally, orally, intranasally, pulmonary, or subcutaneously.
  • the prophylactic or therapeutic agents may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents Administration can be systemic or local.
  • the prophylactic or therapeutic agents disclosed herein may be desirable to administer the prophylactic or therapeutic agents disclosed herein locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous or non-porous material, including membranes and matrices, such as sialastic membranes, polymers, fibrous matrices (e.g., Tissuel®), or collagen matrices.
  • an effective amount of one or more binding proteins disclosed herein antagonists is administered locally to the affected area to a subject to prevent, treat, manage, and/or ameliorate a disorder or a symptom thereof.
  • an effective amount of one or more binding proteins disclosed herein is administered locally to the affected area in combination with an effective amount of one or more therapies (e.g., one or more prophylactic or therapeutic agents) other than an antibody disclosed herein of a subject to prevent, treat, manage, and/or ameliorate a disorder or one or more symptoms thereof.
  • therapies e.g., one or more prophylactic or therapeutic agents
  • the nucleic acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.
  • a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.
  • the method disclosed herein may comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion).
  • Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
  • compositions formulated as depot preparations may additionally comprise of administration of compositions formulated as depot preparations.
  • long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection.
  • the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
  • compositions formulated as neutral or salt forms include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • one or more of the prophylactic or therapeutic agents, or pharmaceutical compositions disclosed herein is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent.
  • one or more of the prophylactic or therapeutic agents, or pharmaceutical compositions disclosed herein is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject.
  • one or more of the prophylactic or therapeutic agents or pharmaceutical compositions disclosed herein is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg.
  • the lyophilized prophylactic or therapeutic agents or pharmaceutical compositions disclosed herein should be stored at between 2° C. and 8° C.
  • the prophylactic or therapeutic agents, or pharmaceutical compositions disclosed herein should be administered within 1 week, within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted.
  • one or more of the prophylactic or therapeutic agents or pharmaceutical compositions disclosed herein is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent.
  • the liquid form of the administered composition is supplied in a hermetically sealed container at least 0.25 mg/ml, at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml.
  • the liquid form should be stored at between 2° C. and 8° C. in its original container.
  • binding proteins and binding protein-portions disclosed herein can be incorporated into a pharmaceutical composition suitable for parenteral administration.
  • the binding protein or binding protein-portions will be prepared as an injectable solution containing 0.1-250 mg/ml binding protein.
  • the injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampoule or pre-filled syringe.
  • the buffer can be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0).
  • Other suitable buffers include but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate.
  • Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for a liquid dosage form).
  • Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%).
  • Other suitable cryoprotectants include trehalose and lactose.
  • Bulking agents can be included for a lyophilized dosage form, principally 1-10% mannitol (optimally 2-4%).
  • Stabilizers can be used in both liquid and lyophilized dosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM).
  • Other suitable bulking agents include glycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).
  • Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants.
  • compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies.
  • Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e., binding protein or binding protein portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile lyophilized powders for the preparation of sterile injectable solutions
  • the methods of preparation include vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including, in the composition, an agent that delays absorption, for example, monostearate salts and gelatin.
  • the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • Supplementary active compounds can also be incorporated into the compositions.
  • a binding protein or binding protein portion disclosed herein is coformulated with and/or coadministered with one or more additional therapeutic agents that are useful for treating disorders in which TNF- ⁇ activity is detrimental.
  • an anti-hTNF- ⁇ antibody or antibody portion disclosed herein may be coformulated and/or coadministered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules).
  • one or more binding proteins disclosed herein may be used in combination with two or more of the foregoing therapeutic agents.
  • Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
  • a binding protein to TNF- ⁇ or fragment thereof is linked to a half-life extending vehicle known in the art.
  • vehicles include, but are not limited to, the Fc domain, polyethylene glycol, and dextran.
  • Such vehicles are described, e.g., in U.S. Pat. No. 6,660,843.
  • nucleic acid sequences comprising nucleotide sequences encoding a binding protein disclosed herein or another prophylactic or therapeutic agent disclosed herein are administered to treat, prevent, manage, or ameliorate a disorder or one or more symptoms thereof by way of gene therapy.
  • Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid.
  • the nucleic acids produce their encoded binding protein or prophylactic or therapeutic agent disclosed herein that mediates a prophylactic or therapeutic effect.
  • TNF- ⁇ plays a critical role in the pathology associated with a variety of diseases involving immune and inflammatory elements, such as autoimmune diseases, particularly those associated with inflammation, including Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, systemic lupus erythematosus, and ankylosing spondylitis. Therefore, the binding proteins herein may be used to treat these disorders.
  • autoimmune diseases particularly those associated with inflammation, including Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, systemic lupus erythematosus, and ankylosing spondylitis. Therefore, the binding proteins herein may be used
  • the disorder is a respiratory disorder; asthma; allergic and nonallergic asthma; asthma due to infection; asthma due to infection with respiratory syncytial virus (RSV); chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation; eosinophilia; fibrosis and excess mucus production; cystic fibrosis; pulmonary fibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema; allergic rhinitis; allergic enterogastritis; an inflammatory and/or autoimmune condition of the skin; an inflammatory and/or autoimmune condition of gastrointestinal organs; inflammatory bowel diseases (IBD); ulcerative colitis; an inflammatory and/or autoimmune condition of the liver; liver cirrhosis; liver fibrosis; liver fibrosis caused by hepatitis B and/or C virus; scleroderma; tumors or cancers; hepatocellular carcinoma; glioblastoma; lymph
  • VH variable heavy
  • VL variable light
  • the AE11-5 human antibody to human TNF was affinity matured by in vitro display technology.
  • One light chain library was constructed to contain limited mutagenesis at the following residues: 28, 31, 32, 51, 55, 91, 92, 93, 95a and 96 (Kabat numbering).
  • This library also contained framework germline back-mutations D1E, M4L, H11Q, R49K, H76N and Q103K as well as toggled residues at position 50(R/K) and 94(S/L) to allow for framework germlining during library selections.
  • CDRH1 and CDRH2 Two heavy chain libraries were made to contain limited mutagenesis in CDRH1 and CDRH2 at residues 30, 31, 33, 50, 52, and 55 to 58 (Kabat numbering) or in CDRH3 at residues 95 to 100b.
  • the library containing CDRH1 and CDRH2 diversities also had framework germline back-mutations A18V and L64Q and toggled residue at 54(L/F) and 78(V/A).
  • the CDRH3 library has an additional toggled residue at 100c(A/F).
  • Table 11 provides a list of amino acid sequences of VH regions of affinity matured fully human TNF antibodies derived from AE11-5 Amino acid residues of individual CDRs of each VH sequence are indicated in bold.
  • Table 12 provides a list of amino acid sequences of VL regions of affinity matured fully human TNF antibodies derived from AE11-5 Amino acid residues of individual CDRs of each VH sequence are indicated in bold.
  • the plate was washed 3 times with PBS/Tween. 50 ⁇ l of streptavidin-HRP diluted 1:10,000 in PBS/0.1% BSA was added to appropriate wells and incubated for 1 hour at RT. The plate was washed 3 times with PBS/Tween. 50 ⁇ l of TMB was added to appropriate wells and the reaction was allowed to proceed for 1 minute. The reaction was stopped with 50 ⁇ l/well 2N H 2 SO 4 and the absorbance read at 450 nm. Results are shown in Table 17.
  • Human TNF was prepared at Abbott Bioresearch Center (Worcester, Mass., US) and received from the Biologics Pharmacy. Mouse TNF was prepared at Abbott Bioresearch Center and received from the Biologics Pharmacy. Rat TNF was prepared at Abbott Bioresearch Center and received from the Biologics Pharmacy. Rabbit TNF was purchased from R&D Systems. Rhesus/Macaque TNF (rhTNF) was purchased from R&D Systems. Actinomycin was purchased from Sigma Aldrich and resuspended at a stock concentration of 10 mg/mL in DMSO.
  • Assay Media 10% FBS (Hyclone#SH30070.03), Gibco reagents: RPMI 1640 (#21870), 2 mM L-glutamine (#25030), 50 units/mL penicillin/50 ⁇ g/mL streptomycin (#15140), 0.1 mM MEM non-essential amino acids (#11140) and 5.5 ⁇ 10 ⁇ 5 M 2-mercaptoethanol (#21985-023).
  • L929 cells were grown to a semi-confluent density and harvested using 0.05% tryspin (Gibco#25300). The cells were washed with PBS, counted, and resuspended at 1E6 cells/mL in assay media containing 4 ⁇ g/mL actinomycin D. The cells were seeded in a 96-well plate (Costar#3599) at a volume of 50 ⁇ L and 5E4 cells/well. Wells received 50 ⁇ L of assay media, bringing the volume to 100 ⁇ L.
  • test sample was prepared as follows. The test and control IgG proteins were diluted to a 4 ⁇ concentration in assay media and serial 1:3 dilutions were performed. TNF species were diluted to the following concentrations in assay media: 400 pg/mL huTNF, 200 pg/mL muTNF, 600 pg/mL ratTNF, and 100 pg/mL rabTNF. Antibody sample (200 ⁇ L) was added to the TNF (200 ⁇ L) in a 1:2 dilution scheme and allowed to incubate for 0.5 hour at room temperature.
  • the antibody/TNF solution was added to the plated cells at 100 ⁇ L for a final concentration at 375 nM-0.019 nM.
  • the final concentration of TNF was as follows: 100 pg/mL huTNF, 50 pg/mL muTNF, 150 pg/mL ratTNF, and 25 pg/mL rabTNF.
  • the plates were incubated for 20 hours at 37° C., 5% CO 2 .
  • 100 ⁇ L was removed from the wells and 10 ⁇ L of WST-1 reagent (Roche cat#11644807001) was added.
  • the mouse anti-human TNF antibody MAK-195 was humanized and affinity-matured to generate a panel of humanized MAK195 variants that have cross-reactivity to cyno-TNF and improved affinity and binding kinetics against both human and cyno TNF.
  • hMAK195 hypermutated CDR residues were identified from other human antibody sequences in the IgBLAST database that also shared high identity to germlines VH3-53 and IGKV1-39. The corresponding hMAK195 CDR residues were then subjected to limited mutagenesis by PCR with primers having low degeneracy at these positions to create three antibody libraries in the scFv format.
  • the first library contained mutations at residues 31, 32, 33, 35, 50, 52, 53, 54, 56 and 58 in the VH CDR1 and 2 (Kabat numbering); the second library at residues 95 to 100, 100a, 101, and 102 in VH CDR3; and the third library at residues 28, 30, 31, 32, 50, 53, 92, 93, 94, and 95 in the three VL CDRs.
  • a binary degeneracy at VH positions 60 (D/A), 61 (S/D), 62 (T/S), 63 (L/V), and 65 (S/G) were introduced into the first library.
  • hMAK195 variants were selected against a low concentration of biotinylated TNF for improved on-rate, off-rate, or both were carried out and antibody protein sequences of affinity-modulated hMAK195 were recovered for converting back to IgG for further characterization. All three libraries were selected separately for the ability to bind human or cynomolgus monkey TNF in the presence of decreasing concentrations of biotinylated human or cynomolgus monkey TNF antigens. All mutated CDR sequences recovered from library selections were recombined into additional libraries and the recombined libraries were subjected to more stringent selection conditions before individual antibodies are identified.
  • Table 19 provides a list of amino acid sequences of VH and VL of the humanized MAK-195 which were subjected to the affinity maturation selection protocol Amino acid residues of individual CDRs of each VH and VL sequence are indicated in bold.
  • Table 20 provides a list of amino acid sequences of VL regions of affinity matured fully human TNF antibodies derived from hMAK195 Amino acid residues of individual CDRs of each VH sequence are indicated in bold.
  • hMAK195 Heavy chain variable region (SEQ ID NO: 1075) hMAK195VH EVQLVESGGGLVQPGGSLRLSCAASGFTFS DYGVN WVRQAPGKGLEWVS MIWGDGSTD NFS T I RAG T A HLN S V GSE F H YS H L SDA A V IR Q R AEV Y S Y K LVG W N S NY G YDSTLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREWHHGPVAYWGQGTLVTVSS ADSV G HSQQRTLDS QLRPASGVF LCLLVQDGC YRYNWAETN DFPYEKW P NDARS R I TYVTP P H PPDDI A AICA I SG C R hMAK195 Light chain variable region (SEQ ID NO: 1076) hMAK195VL DIQMTQSPSSLSASVGDRVTITC
  • the mouse anti-human TNF antibody MAK-199 was humanized and affinity-matured to generate a panel of humanized MAK195 variants that have improved affinity and binding kinetics against both human and cyno TNF.
  • Several libraries were made according to specifications below:
  • H1+H2 (DDK) library Three HC libraries were made after the V2I back-mutation was first introduced and confirmed that it did not impact scFv affinity to TNF.
  • Germline toggles T51A, Y71F, F87Y, and T43A/V44P (these two co-evolve)
  • VH libraries will be recombined with and without VL library after library diversity is reduced after at least 3 rounds of selection.
  • Table 27 provides a list of amino acid sequences of VH of the hMAK-199 antibody which were subjected to the affinity maturation selection protocol Amino acid residues of individual CDRs of each VH sequence are indicated in bold.
  • Table 28 provides a list of amino acid sequences of VL regions of affinity matured fully human TNF antibodies derived from hMAK199 Amino acid residues of individual CDRs of each VL sequence are indicated in bold.
  • the BIACORE assay (Biacore, Inc. Piscataway, N.J.) determines the affinity of binding proteins with kinetic measurements of on-rate and off-rate constants. Binding of binding proteins to a target antigen (for example, a purified recombinant target antigen) is determined by surface plasmon resonance-based measurements with a Biacore® 1000 or 3000 instrument (Biacore®AB, Uppsala, Sweden) using running HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25° C.
  • a target antigen for example, a purified recombinant target antigen
  • HBS-EP 10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20
  • Unmodified carboxymethyl dextran without goat anti-mouse IgG in flow cell 1 and 3 is used as the reference surface.
  • rate equations derived from the 1:1 Langmuir binding model are fitted simultaneously to association and dissociation phases of all eight injections (using global fit analysis) with the use of Biaevaluation 4.0.1 software.
  • Purified antibodies are diluted in HEPES-buffered saline for capture across goat anti-mouse IgG specific reaction surfaces.
  • Antibodies to be captured as a ligand 25 ⁇ g/ml are injected over reaction matrices at a flow rate of 5 ⁇ l/minute.
  • binding proteins herein are expected to have beneficial properties in this regard, including high affinity, slow off rate, and high neutralizing capacity.
  • L929 cells are grown to a semi-confluent density and harvested using 0.25% trypsin
  • the cells are washed with PBS, counted and resuspended at 1E6 cells/mL in assay media containing 4 ⁇ g/mL actinomycin D.
  • the cells are seeded in a 96-well plate (Costar#3599) at a volume of 100 ⁇ L and 5E4 cells/well.
  • the binding proteins and control IgG are diluted to a 4 ⁇ concentration in assay media and serial 1:4 dilutions are performed.
  • the huTNF- ⁇ is diluted to 400 pg/mL in assay media.
  • Binding protein sample 200 ⁇ L
  • Binding protein sample 200 ⁇ L
  • huTNF- ⁇ 200 ⁇ L
  • a 1:2 dilution scheme allowed to incubate for 0.5 hour at room temperature.
  • the binding protein/human TNF- ⁇ solution is added to the plated cells at 100 ⁇ L for a final concentration of 100 pg/mL huTNF- ⁇ and 150 nM-0.0001 nM binding protein.
  • the plates are incubated for 20 hours at 37° C., 5% CO 2 .
  • 100 ⁇ L is removed from the wells and 10 ⁇ L of WST-1 reagent (Roche cat#11644807001) is added. Plates are incubated under assay conditions for 3.5 hours. The plates are read at OD 420-600 nm on a Spectromax 190 ELISA plate reader.
  • binding proteins herein are expected to have beneficial properties in this regard, including high affinity, slow off rate, and high neutralizing capacity.
  • a patient requiring treatment with a TNF- ⁇ binding protein may have a disease with immune and inflammatory elements, such as autoimmune diseases, particularly those associated with inflammation, including Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, and ankylosing spondylitis. Therefore, the binding proteins herein may be used to treat these disorders.
  • autoimmune diseases particularly those associated with inflammation, including Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, and ankylosing spondylitis. Therefore, the binding proteins herein may be used to treat these disorders.
  • TNF- ⁇ binding protein may occur by subcutaneous injection. If the patient has rheumatoid arthritis, psoratic arthritis, or ankylosing spondyitis, the patient may receive 40 mg every other week as a starting dose and 40 mg every week, if necessary to achieve treatment goals. If the patient has juvenile idiopathic arthritis and weighs from 15 kg to ⁇ 30 kg, the patient may receive 20 mg every other week, and if >30 kg, 40 mg every other week. If the patient has Crohn's disease, the patient may receive an initial dose of 160 mg (four 40 mg injections in one day or two 40 mg injections per day for two consecutive days) followed by 80 mg two weeks later, and another two weeks later begin a maintenance dose of 40 mg every other week. If the patient has plaque psoriasis, the patient may receive an 80 mg initial dose, followed by 40 mg every other week starting one week after initial dose.
  • the binding protein may be provided in a single-use prefilled pen (40 mg/0.8 mL), a single-use prefilled glass syringe (40 mg/0.8 mL or 20 mg/0.4 mL).

Abstract

Isolated binding proteins, e.g., antibodies or antigen binding portions thereof, which bind to tumor necrosis factor-alpha (TNF-α), e.g., human TNF-α, and related antibody-based compositions and molecules are disclosed. Also disclosed are pharmaceutical compositions comprising the antibodies, as well as therapeutic and diagnostic methods for using the antibodies.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application claims priority to U.S. Provisional Application Ser. No. 61/550,587, filed Oct. 24, 2011, which is incorporated herein by reference in its entirety.
    • BACKGROUND OF THE INVENTION
  • 1. Field of the Invention
  • TNF-α binding proteins and their uses in the prevention and/or treatment of acute and chronic immunological diseases are provided.
  • 2. Background of the Invention
  • There is a need in the art for improved binding proteins capable of binding TNF-α (also referred to as tumor necrosis factor, tumor necrosis factor-alpha, tumor necrosis factor-α, TNF, and cachectin). Provided are a novel family of binding proteins, CDR grafted binding proteins, humanized binding proteins, and fragments thereof, capable of binding TNF-α with high affinity and neutralizing TNF-α.
    • BRIEF SUMMARY OF THE INVENTION
  • TNF-α binding proteins, or antigen-binding portions thereof, that bind TNF-α are provided. In an embodiment, the antigen binding domain comprises the VH region chosen from any one of SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34-58, 74-83, 94-266, 478-486, 496-675, 738-762, 778-956, 1053-1062, 1073, 1075, and 1077, or one, two, or three CDRs therefrom. In another embodiment, the antigen binding domain comprises the VL region chosen from any one of SEQ ID NOs: 23, 25, 27, 29, 31, 33, 59-73, 84-93, 267-477, 487-495, 676-737, 763-777, 957-1052, 1063-1072, 1074, 1076, and 1078, or one, two, or three CDRs therefrom. In a particular embodiment, the antigen binding domain comprises a VH region and a VL region, for example, wherein the VH region comprises SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34-58, 74-83, 94-266, 478-486, 496-675, 738-762, 778-956, 1053-1062, 1073, 1075, and 1077, or one, two, or three CDRs therefrom, and the VL region comprises SEQ ID NOs: 23, 25, 27, 29, 31, 33, 59-73, 84-93, 267-477, 487-495, 676-737, 763-777, 957-1052, 1063-1072, 1074, 1076, and 1078, or one, two, or three CDRs therefrom.
  • In an embodiment, the binding protein binds TNF-α. In another embodiment, the binding protein modulates a biological function of TNF-α. In another embodiment, the binding protein neutralizes TNF-α. In yet another embodiment, the binding protein diminishes the ability of TNF-α to bind to its receptor, for example, the binding protein diminishes the ability of pro-human TNF-α, mature-human TNF-α, or truncated-human TNF-α to bind to its receptor. In yet another embodiment, the binding protein reduces one or more TNF-α biological activities selected from: TNF-dependent cytokine production; TNF-dependent cell killing; TNF-dependent inflammation; TNF-dependent bone erosion; and TNF-dependent cartilage damage.
  • In an embodiment, the binding protein has an on rate constant (Kon) selected from: at least about 102M−1 s−1; at least about 103M−1 s−1; at least about 104M−1 s−1; at least about 105M−1 s−1; and at least about 106M−1 s−1; as measured by surface plasmon resonance. In another embodiment, the binding protein has an off rate constant (Koff) selected from: at most about 10−3 s−1; at most about 10−4 s−1; at most about 10−5 s−1; and at most about 10−6 s−1, as measured by surface plasmon resonance. In yet another embodiment, the binding protein has a dissociation constant (KD) selected from: at most about 10−7 M; at most about 10−8 M; at most about 10−9 M; at most about 10−10 M; m at most about 10−11 M; at most about 10−12 M; and at most 10−13M.
  • In another aspect, a method for treating a mammal is provided comprising administering to the mammal an effective amount of the pharmaceutical composition disclosed herein. In another embodiment, a method for reducing human TNF-α activity is provided, the method comprising: contacting human TNF-α with the binding protein disclosed herein such that human TNF-α activity is reduced. In another embodiment, provided is a method for reducing human TNF-α activity in a human subject suffering from a disorder in which TNF-α activity is detrimental, the method comprising administering to the human subject the binding protein disclosed herein such that human TNF-α activity in the human subject is reduced. In another embodiment, provided is a method for treating a subject for a disease or a disorder in which TNF-α activity is detrimental, the method comprising administering to the subject the binding protein disclosed herein such that treatment is achieved.
  • In one embodiment, the method treats diseases involving immune and inflammatory elements, such as autoimmune diseases, particularly those associated with inflammation, including Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoriatic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, and ankylosing spondylitis. Therefore, the binding proteins herein may be used to treat these disorders.
    • DETAILED DESCRIPTION OF THE INVENTION
  • Provided are TNF-α binding proteins, or antigen-binding portions thereof, that bind TNF-α, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such binding proteins and fragments. Also provided are methods of using the binding proteins disclosed herein to detect human TNF-α, to inhibit human TNF-α either in vitro or in vivo, and to regulate gene expression or TNF-α related functions.
  • Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and/or”, unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms of the term, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
  • Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
  • The term “human TNF-α” (abbreviated herein as hTNF-α) includes a trimeric cytokine protein. The term includes a homotrimeric protein comprising three 17.5 kD TNF-α proteins. The homotrimeric protein is referred to as a “TNF-α protein”. The term human “TNF-α” is intended to include recombinant human TNF-α (rhTNF-α), which can be prepared by standard recombinant expression methods. The sequence of human TNF-α is shown in Table 1.
  • TABLE 1
    Sequence of Human TNF-α
    Sequence
    Identi- Sequence
    Protein fier 12345678901234567890123456789012
    Human SEQ VRSSSRTPSDKPVAHVVANPQAEGQLQWLNDR
    TNF-α ID NO.: 1 ANALLANGVELRDNQLVVPSEGLYLIYSQVLF
    KGQGCPSTHVLLTHTISRIAVSYQTKVNLLSA
    IKSPCQRETPEGAEAKPWYEPIYLGGVFQLEK
    GDRLSAEINRPDYLDFAESGQVYFGIIAL
  • The term “antibody”, broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.
  • In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.
  • The term “antigen-binding portion” or “antigen-binding region” of a binding protein (or simply “binding protein portion”), refers to one or more fragments of a binding protein that retain the ability to specifically bind to an antigen (e.g., hTNF-α). The antigen-binding function of a binding protein can be performed by fragments of a full-length binding protein. Such binding protein embodiments may also have bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen-binding portion” of a binding protein include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature 341:544-546, Winter et al., PCT publication WO 90/05144 A1), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain binding proteins are also intended to be encompassed within the term “antigen-binding portion” of a binding protein. Other forms of single chain binding proteins, such as diabodies are also encompassed. Diabodies are bivalent, bispecific binding proteins in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, e.g., Holliger, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, et al. (1994) Structure 2:1121-1123).
  • The term “binding protein” refers to a polypeptide comprising one or more antigen-binding portions disclosed herein optionally linked to a linker polypeptide or a constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see e.g., Holliger, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, et al. (1994) Structure 2:1121-1123). A constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art and represented in Table 2.
  • TABLE 2
    Sequence of Human IgG Heavy Chain Constant Domain and Light
    Chain Constant Domain
    Sequence Sequence
    Protein Identifier 12345678901234567890123456789012
    Ig gamma-1 SEQ ID NO.: 2 ASTKGPSVFFLAPSSKSTSGGTAALGCLVKDY
    constant region FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
    LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
    KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
    KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
    YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
    HQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
    QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
    PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
    LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
    QKSLSLSPGK
    Ig gamma-1 SEQ ID NO.: 3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
    constant region FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
    mutant LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
    KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP
    KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
    YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
    HQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
    QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
    PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
    LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
    QKSLSLSPGK
    Ig Kappa constant SEQ ID NO.: 4 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY
    region PREAKVQWKVDNALQSGNSQESVTEQDSKDST
    YSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
    VTKSFNRGEC
    Ig Lambda SEQ ID NO.: 5 QPKAAPSVTLFPPSSEELQANKATLVCLISDF
    constant region YPGAVTVAWKADSSPVKAGVETTTPSKQSNNK
    YAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE
    KTVAPTECS
  • A binding protein, or antigen-binding portion thereof, may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the binding protein or binding protein portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, et al. (1995) Hum. Antibod. Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, binding proteins, binding protein portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
  • An “isolated binding protein” refers to a binding protein, or antigen-binding portion thereof, that is substantially free of other binding proteins having different antigenic specificities (e.g., an isolated binding protein that specifically binds hTNF-α is substantially free of binding proteins that specifically bind antigens other than hTNF-α). An isolated binding protein that specifically binds hTNF-α may, however, have cross-reactivity to other antigens, such as TNF-α molecules from other species. Moreover, an isolated binding protein may be substantially free of other cellular material and/or chemicals.
  • The term “human binding protein” includes binding proteins, or antigen-binding portion thereof, that having variable and constant regions derived from human germline immunoglobulin sequences. The human binding proteins disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human binding protein”, is not intended to include binding proteins in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). See also, Martin, “Protein Sequence and Structure Analysis of Antibody Variable Domains,” In Kontermann and Dübel, eds., Antibody Engineering (Springer-Verlag, Berlin, 2001), Chapter 31, especially pages 432-433. For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 106 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
  • The term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “CDR set” refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917) and Chothia et al. (1989) Nature 342:877-883) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (1995) FASEB J. 9:133-139 and MacCallum (1996) J. Mol. Biol. 262(5):732-745. Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although particular embodiments use Kabat or Chothia defined CDRs.
  • Human heavy chain and light chain acceptor sequences are known in the art. In one embodiment of the disclosure the human heavy chain and light chain acceptor sequences are selected from the sequences listed from V-base (hvbase.mrc-cpe.cam.ac.uk/) or from IMGT®, the international ImMunoGeneTics information system® (himgt.cines.fr/textes/IMGTrepertoire/LocusGenes/). In another embodiment of the disclosure the human heavy chain and light chain acceptor sequences are selected from the sequences described in Table 3 and Table 4, respectively.
  • TABLE 3
    Heavy Chain Acceptor Sequences
    SEQ ID Protein Sequence
    No. region 12345678901234567890123456789012
    SEQ ID VH4-59 FR1 QVQLQESGPGLVKPSETLSLTCTVSGGSISS
    NO: 6
    SEQ ID VH4-59 FR2 WIRQPPGKGLEWIG
    NO: 7
    SEQ ID VH4-59 FR3 RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
    NO: 8
    SEQ ID VH3-53 FR1 EVQLVESGGGLIQPGGSLRLSCAASGFTVSS
    NO: 9
    SEQ ID VH3-53 FR2 WVRQAPGKGLEWVS
    NO: 10
    SEQ ID VH3-53 FR3 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
    NO: 11
    SEQ ID JH1/JH4/JH5 WGQGTLVTVSS
    NO: 12 FR4
    SEQ ID JH2 FR4 WGRGTLVTVSS
    NO: 13
    SEQ ID JH6 FR4 WGQGTTVTVSS
    NO: 14
  • TABLE 4
    Light Chain Acceptor Sequences
    SEQ ID Protein Sequence
    No. region 12345678901234567890123456789012
    SEQ ID 1-39/O12 DIQMTQSPSSLSASVGDRVTITC
    NO: 15 FR1
    SEQ ID 1-39/O12 WYQQKPGKAPKLLIY
    NO: 16 FR2
    SEQ ID 1-39/O12 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
    NO: 17 FR3
    SEQ ID 3-15/L2 FR1 EIVMTQSPATLSVSPGERATLSC
    NO: 18
    SEQ ID 3-15/L2 FR2 WYQQKPGQAPRLLIY
    NO: 19
    SEQ ID 3-15/L2 FR3 GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC
    NO: 20
    SEQ ID JK2 FR4 FGQGTKLEIKR
    NO: 21
  • The term “multivalent binding protein” is used in this specification to denote a binding protein comprising two or more antigen binding sites. The multivalent binding protein may be engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody. The term “multispecific binding protein” refers to a binding protein capable of binding two or more related or unrelated targets. Dual variable domain (DVD) binding proteins or immunoglobulins (DVD-Ig) as used herein, are binding proteins that comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. Such DVD-binding proteins may be monospecific, i.e., capable of binding one antigen or multispecific, i.e., capable of binding two or more antigens. DVD-binding proteins comprising two heavy chain DVD-Ig polypeptides and two light chain DVD-Ig polypeptides are referred to a DVD-Ig. Each half of a DVD-Ig comprises a heavy chain DVD-Ig polypeptide, and a light chain DVD-Ig polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site. DVD binding proteins and methods of making DVD binding proteins are disclosed in U.S. Pat. No. 7,612,181.
  • One aspect of the disclosure pertains to a DVD binding protein comprising binding proteins capable of binding TNF-α. In a particular embodiment, the DVD binding protein is capable of binding TNF-α and a second target.
  • The term “neutralizing” refers to neutralization of a biological activity of a cytokine when a binding protein specifically binds the cytokine. In a particular embodiment, binding of a neutralizing binding protein to hTNF-α results in inhibition of a biological activity of hTNF-α, e.g., the neutralizing binding protein binds hTNF-α and reduces a biologically activity of hTNF-α by at least about 20%, 40%, 60%, 80%, 85% or more Inhibition of a biological activity of hTNF-α by a neutralizing binding protein can be assessed by measuring one or more indicators of hTNF-α biological activity well known in the art. For example neutralization of the cytoxicity of TNF-α on L929 cells.
  • In another embodiment, the terms “agonist” or “agonizing” refer to an increase of a biological activity of TNF-α when a binding protein specifically binds TNF-α, e.g., hTNF-α. In a particular embodiment, binding of an agonizing binding protein to TNF-α results in the increase of a biological activity of TNF-α. In a particular embodiment, the agonistic binding protein binds TNF-α and increases a biologically activity of TNF-α by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95, 96%, 97%, 98%, 99%, and 100%. An inhibition of a biological activity of TNF-α by an agonistic binding protein can be assessed by measuring one or more indicators of TNF-α biological activity well known in the art.
  • The term “activity” includes activities such as the binding specificity/affinity of a binding protein for an antigen, for example, a hTNF-α binding protein that binds to a TNF-α antigen and/or the neutralizing potency (or agonizing potency) of a binding protein, for example, a hTNF-α binding protein whose binding to hTNF-α inhibits the biological activity of hTNF-α, e.g., neutralization of the cytoxicity of TNF-α on L929 cells.
  • The term “surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).
  • The term “Kon” refers to the on rate constant for association of a binding protein (e.g., an antibody) to the antigen to form, e.g., the antibody/antigen complex as is known in the art. The “Kon” also is known by the terms “association rate constant”, or “ka”, as used interchangeably herein. This value indicating the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen also is shown by the equation below:

  • Antibody(“Ab”)+Antigen(“Ag”)→Ab−Ag
  • The term “Koff” refers to the off rate constant for dissociation, or “dissociation rate constant”, of a binding protein (e.g., an antibody), from the, e.g., antibody/antigen complex as is known in the art. This value indicates the dissociation rate of an antibody from its target antigen or separation of Ab−Ag complex over time into free antibody and antigen as shown by the equation below:

  • Ab+Ag←Ab−Ag
  • The term “KD” refers to the “equilibrium dissociation constant” and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). The association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay can be used (e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used.
  • I. Binding Proteins that Bind Human TNF-α
  • One aspect of the present disclosure provides isolated fully-human anti-human TNF binding proteins, such as monoclonal antibodies, or antigen-binding portions thereof, that bind to TNF-α with high affinity, a slow off rate and high neutralizing capacity. A second aspect of the disclosure provides affinity-matured fully-human anti-TNF binding proteins, such as monoclonal antibodies, or antigen-binding portions thereof, that bind to TNF-α with high affinity, a slow off rate and high neutralizing capacity.
  • A. Method of Making TNF-α Binding Proteins
  • The binding proteins disclosed herein may be made by any of a number of techniques known in the art.
  • 1. Anti-TNF-α Monoclonal Antibodies Using Transgenic Animals
  • In another embodiment of the disclosure, binding proteins are produced by immunizing a non-human animal comprising some, or all, of the human immunoglobulin locus with a TNF-α antigen. In a particular embodiment, the non-human animal is a XENOMOUSE transgenic mouse, an engineered mouse strain that comprises large fragments of the human immunoglobulin loci and is deficient in mouse antibody production. See, e.g., Green et al. (1994) Nature Genet. 7:13-21 and U.S. Pat. Nos. 5,916,771; 5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598 and 6,130,364. See also PCT Publications WO 91/10741, published Jul. 25, 1991; WO 94/02602, published Feb. 3, 1994; WO 96/34096 and WO 96/33735, both published Oct. 31, 1996; WO 98/16654, published Apr. 23, 1998; WO 98/24893, published Jun. 11, 1998; WO 98/50433, published Nov. 12, 1998; WO 99/45031, published Sep. 10, 1999; WO 99/53049, published Oct. 21, 1999; WO 00/09560, published Feb. 24, 2000; and WO 00/37504, published Jun. 29, 2000. The XENOMOUSE transgenic mouse produces an adult-like human repertoire of fully human antibodies, and generates antigen-specific human Mabs. The XENOMOUSE transgenic mouse contains approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and x light chain loci. See, Mendez et al. (1997) Nature Genet. 15:146-156; Green and Jakobovits (1998) J. Exp. Med. 188:483-495.
  • 2. Anti-TNF-α Monoclonal Antibodies Using Recombinant Antibody Libraries
  • In vitro methods also can be used to make the binding protein disclosed herein, wherein an antibody library is screened to identify an antibody having the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, U.S. Pat. No. 5,223,409; PCT Publications WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 97/29131; Fuchs et al. (1991) Bio/Technology 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al. (1990) Nature 348:552-554; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nucl. Acid Res. 19:4133-4137; and Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and U.S. Patent Publication No. 2003.0186374.
  • The recombinant antibody library may be from a subject immunized with TNF-α, or a portion of TNF-α. Alternatively, the recombinant antibody library may be from a naïve subject, i.e., one who has not been immunized with TNF-α, such as a human antibody library from a human subject who has not been immunized with human TNF-α. Antibodies disclosed herein are selected by screening the recombinant antibody library with the peptide comprising human TNF-α to thereby select those antibodies that recognize TNF-α. Methods for conducting such screening and selection are well known in the art, such as described in the references in the preceding paragraph. To select antibodies disclosed herein having particular binding affinities for hTNF-α, such as those that dissociate from human TNF-α with a particular koff rate constant, the art-known method of surface plasmon resonance can be used to select antibodies having the desired koff rate constant. To select antibodies disclosed herein having a particular neutralizing activity for hTNF-α, such as those with a particular an IC50, standard methods known in the art for assessing the inhibition of hTNF-α activity may be used.
  • In one aspect, provided is an isolated binding protein, or an antigen-binding portion thereof, that binds TNF-α, e.g., human TNF-α. In a particular embodiment, the binding protein is a neutralizing binding protein. In various embodiments, the binding protein is a recombinant binding protein or a monoclonal antibody.
  • For example, the binding proteins disclosed herein can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the binding proteins disclosed herein can be found in the art.
  • As described in the above references, after phage selection, the binding protein coding regions from the phage can be isolated and used to generate whole binding proteins including human binding protein or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT Publication WO 92/22324; Mullinax et al. (1992) BioTechniques 12(6):864-869; and Sawai et al. (1995) Am. J. Reprod. Immunol. 34:26-34; and Better et al. (1998) Science 240:1041-1043. Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al. (1991) Methods Enzymol. 203:46-88; Shu et al. (1993) Proc. Natl. Acad. Sci. USA 90:7995-7999; and Skerra et al. (1998) Science 240:1038-1041.
  • Alternative to screening of recombinant antibody libraries by phage display, other methodologies known in the art for screening large combinatorial libraries can be applied to the identification of dual specificity binding protein disclosed herein. One type of alternative expression system is one in which the recombinant antibody library is expressed as RNA-protein fusions, as described in PCT Publication No. WO 98/31700 and in Roberts and Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this system, a covalent fusion is created between an mRNA and the peptide or protein that it encodes by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end. Thus, a specific mRNA can be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein, e.g., antibody, or portion thereof, such as binding of the antibody, or portion thereof, to the dual specificity antigen. Nucleic acid sequences encoding antibodies, or portions thereof, recovered from screening of such libraries can be expressed by recombinant means as described above (e.g., in mammalian host cells) and, moreover, can be subjected to further affinity maturation by either additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence(s), or by other methods for affinity maturation in vitro of recombinant antibodies, as described above.
  • In another approach the binding proteins disclosed herein can also be generated using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of yeast. In particular, such yeast can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Examples of yeast display methods that can be used to make the binding proteins disclosed herein include those disclosed Wittrup et al. U.S. Pat. No. 6,699,658 and Frenken et al., U.S. Pat. No. 6,114,147.
  • B. Production of Recombinant TNF-α Binding Proteins
  • Binding proteins disclosed herein may be produced by any of a number of techniques known in the art. For example, expression from host cells, wherein expression vector(s) encoding the heavy and light chains is (are) transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is possible to express the binding proteins disclosed herein in either prokaryotic or eukaryotic host cells, expression of binding protein in eukaryotic cells is contemplated, for example, in mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active binding protein.
  • Mammalian host cells for expressing the recombinant binding proteins disclosed herein include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) J. Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding binding protein genes are introduced into mammalian host cells, the binding proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the binding protein in the host cells or, in particular, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
  • Host cells can also be used to produce functional binding protein fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and/or the heavy chain of a binding protein disclosed herein. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the binding proteins disclosed herein. In addition, bifunctional binding proteins may be produced in which one heavy and one light chain are a binding protein disclosed herein and the other heavy and light chain are specific for an antigen other than the antigens of interest by crosslinking a binding protein disclosed herein to a second binding protein by standard chemical crosslinking methods.
  • In an exemplary system for recombinant expression of a binding protein, or antigen-binding portion thereof, disclosed herein, a recombinant expression vector encoding both the heavy chain and the light chain is introduced into dhfr− CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the heavy and light chains and intact binding protein is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the binding protein from the culture medium. Still further a method of synthesizing a recombinant binding protein disclosed herein is provided by culturing a host cell disclosed herein in a suitable culture medium until a recombinant binding protein disclosed herein is synthesized. The method can further comprise isolating the recombinant binding protein from the culture medium.
  • II. hTNF-α Binding Proteins
  • A. Individual Clone Sequences
  • Table 5 provides the VH and VL sequences of fully human anti-human TNF binding proteins, including CDRs from each VH and VL sequence.
  • TABLE 5
    Individual Fully Human Anti-TNF-α VH Sequences
    Sequence
    Protein region 123456789012345678901234567890
    AE11-1 VH SEQ ID NO.: 22 EVQLVQSGAEVKKPGASVKVSCKASGYTFT
    SYDVNWVRQATGQGLEWMGWMNPNSGNTGY
    AQKFQGRVTITADESTSTAYMELSSLRSED
    TAVYYCAIFDSDYMDVWGKGTLVTVSS
    AE11-1 VH CDR- Residues 31-35 SYDVN
    H1 of SEQ ID
    NO.: 22
    AE11-1 VH CDR- Residues 50-66 WMNPNSGNTGYAQKFQG
    H2 of SEQ ID
    NO.: 22
    AE11-1 VH CDR- Residues 99-106 FDSDYMDV
    H3 of SEQ ID
    NO.: 22
    AE11-1 VL SEQ ID NO.: 23 SYELTQPPSVSLSPGQTARITCSGDALPKQ
    YAYWYQQKPGQAPVLVIYKDTERPSGIPER
    FSGSSSGTTVTLTISGAQAEDEADYYCQSA
    DSSGTSWVFGGGTKLTVL
    AE11-1 VL CDR- Residues 23-33 SGDALPKQYAY
    L1 of SEQ ID
    NO.: 23
    AE11-1 VL CDR- Residues 49-55 KDTERPS
    L2 of SEQ ID
    NO.: 23
    AE11-1 VL CDR- Residues 89-98 SADSSGTSWV
    L3 of SEQ ID
    NO.: 23
    AE11-5 VH SEQ ID NO.: 24 EVQLVQSGAEVKKPGSSAKVSCKASGGTFS
    SYAISWVRQAPGQGLEWMGGIIPILGTANY
    AQKFLGRVTITADESTSTVYMELSSLRSED
    TAVYYCARGLYYDPTRADYWGQGTLVTVSS
    AE11-5 VH CDR- Residues 31-35 SYAIS
    H1 of SEQ ID
    NO.: 24
    AE11-5 VH CDR- Residues 50-66 GIIPILGTANYAQKFLG
    H2 of SEQ ID
    NO.: 24
    AE11-5 VH CDR- Residues 99-109 GLYYDPTRADY
    H3 of SEQ ID
    NO.: 24
    AE11-5 VL SEQ ID NO.: 25 DIVMTQSPDFHSVTPKEKVTITCRASQSIG
    SSLHWYQQKPDQSPKLLIRHASQSISGVPS
    RFSGSGSGTDFTLTIHSLEAEDAATYYCHQ
    SSSSPPPTFGQGTQVEIK
    AE11-5 VL CDR- Residues 24-34 RASQSIGSSLH
    L1 of SEQ ID
    NO.: 25
    AE11-5 VL CDR- Residues 50-56 HASQSIS
    L2 of SEQ ID
    NO.: 25
    AE11-5 VL CDR- Residues 89-98 HQSSSSPPPT
    L3 of SEQ ID
    NO.: 25
    TNF-JK1 VH SEQ ID NO.: 26 EVQLVESGGGLVQPGGSLRLSCATSGFTFN
    NYWMSWVRQAPGKGLEWVANINHDESEKYY
    VDSAKGRFTISRDNAEKSLFLQMNSLRAED
    TAVYYCARIIRGRVGFDYYNYAMDVWGQGT
    LVTVSS
    TNF-JK1 VH CDR- Residues 31-35 NYWMS
    H1 of SEQ ID
    NO.: 26
    TNF-JK1 VH CDR- Residues 50-66 NINHDESEKYYVDSAKG
    H2 of SEQ ID
    NO.: 26
    TNF-JK1 VH CDR- Residues 99-115 IIRGRVGFDYYNYAMDV
    H3 of SEQ ID
    NO.: 26
    TNF-JK1 VL SEQ ID NO.: 27 DIRLTQSPSPLSASVGDRVTITCRASQSIG
    NYLNWYQHKPGKAPKLLIYAASSLQSGVPS
    RFSGTGSGTDFTLTISSLQPEDFATYYCQE
    SYSLIFAGGTKVEIK
    TNF-JK1 VL CDR- Residues 24-34 RASQSIGNYLN
    L1 of SEQ ID
    NO.: 27
    TNF-JK1 VL CDR- Residues 50-56 AASSLQS
    L2 of SEQ ID
    NO.: 27
    TNF-JK1 VL CDR- Residues 89-95 QESYSLI
    L3 of SEQ ID
    NO.: 27
    TNF-Y7C VH SEQ ID NO.: 28 EVQLVQSGAEVKKPGASVKVSCKTSGYTFS
    NYDINWVRQPTGQGLEWMGWMDPNNGNTGY
    AQKFVGRVTMTRDTSKTTAYLELSGLKSED
    TAVYYCARSSGSGGTWYKEYFQSWGQGTMV
    TVSS
    TNF-Y7C VH CDR- Residues 31-35 NYDIN
    H1 of SEQ ID
    NO.: 28
    TNF-Y7C VH CDR- Residues 50-66 WMDPNNGNTGYAQKFVG
    H2 of SEQ ID
    NO.: 28
    TNF-Y7C VH CDR- Residues 99-112 KSSGSGGTWYKEYFQS
    H3 of SEQ ID
    NO.: 28
    TNF-Y7C VL SEQ ID NO.: 29 DIVMTQSPLSLPVTPGEPASISCRSSQSLL
    HSNGYNYLDWYLQKPGQFPQLLIYLGSYRA
    SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
    YYCMQRIEFPPGTFGQGTKLGIK
    TNF-Y7C VL CDR- Residues 24-39 RSSQSLLHSNGYNYLD
    L1 of SEQ ID
    NO.: 29
    TNF-Y7C VL CDR- Residues 55-61 LGSYRAS
    L2 of SEQ ID
    NO.: 29
    TNF-Y7C VL CDR- Residues 94-103 MQRIEFPPGT
    L3 of SEQ ID
    NO.: 29
    AE11-7 VH SEQ ID NO.: 30 EVQLVQSGAEVKKPGASVKVSCKTSGYSLT
    QYPIHWVRQAPGQRPEWMGWISPGNGNTKL
    SPKFQGRVTLSRDASAGTVFMDLSGLTSDD
    TAVYFCTSVDLGDHWGQGTLVTVSS
    AE11-7 VH CDR- Residues 31-35 QYPIH
    H1 of SEQ ID
    NO.: 30
    AE11-7 VH CDR- Residues 50-66 WISPGNGNTKLSPKFQG
    H2 of SEQ ID
    NO.: 30
    AE11-7 VH CDR- Residues 99-104 VDLGDH
    H3 of SEQ ID
    NO.: 30
    AE11-7 VL SEQ ID NO.: 31 DIVMTQSPEFQSVTPKEKVTITCRASQSIG
    SSLHWYQQKPDQSPKLLINYASQSFSGVPS
    RFSGGGSGTDFTLTINSLEAEDAATYYCHQ
    SSNLPITFGQGTRLEIK
    AE11-7 VL CDR- Residues 24-34 RASQSIGSSLH
    L1 of SEQ ID
    NO.: 31
    AE11-7 VL CDR- Residues 50-56 YASQSFS
    L2 of SEQ ID
    NO.: 31
    AE11-7 VL CDR- Residues 89-97 HQSSNLPIT
    L3 of SEQ ID
    NO.: 31
    AE11-13 VH SEQ ID NO.: 32 EVQLVESGGGLVQPGRSLRLSCAASGFTFD
    DYPMHWVRQAPGEGLEWVSGISSNSASIGY
    ADSVKGRFTISRDNAQNTLYLQMNSLGDED
    TAVYYCVSLTLGIGQGTLVTVSS
    AE11-13 VH CDR- Residues 31-35 DYPMH
    H1 of SEQ ID
    NO.: 32
    AE11-13 VH CDR- Residues 50-66 GISSNSASIGYADSVKG
    H2 of SEQ ID
    NO.: 332
    AE11-13 VH CDR- Residues 99-102 LTLG
    H3 of SEQ ID
    NO.: 32
    AE11-13 VL SEQ ID NO.: 33 DIRLTQSPSSLSASVGDRVTITCRASQSIG
    NYLHWYQQKPGKAPKLLIYAASSLQSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    SYSTLYSFGQGTKLEIK
    AE11-13 VL CDR- Residues 24-34 RASQSIGNYLH
    L1 of SEQ ID
    NO.: 33
    AE11-13 VL CDR- Residues 50-56 AASSLQS
    L2 of SEQ ID
    NO.: 33
    AE11-13 VL CDR- Residues 89-97 QQSYSTLYS
    L3 of SEQ ID
    NO.: 33
  • B. IgG Converted Clones
  • Table 6 provides the VH sequence of humanized anti-TNF MAK-195 antibodies that were converted into IgG clones as discussed in detail in Example 2.
  • TABLE 6
    Humanized anti-TNF MAK-195 Ab VH sequences of IgG
    converted clones
    Protein Sequence
    region 123456789012345678901234567890
    A8 SEQ ID NO.: 34 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVNWVRQAPGKGLEWVSMIAADGFTDYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWHHGPVAYWGQGTLVTVSS
    A8 CDR-H1 Residues 31-35 NYGVN
    VH of SEQ ID
    NO.: 34
    A8 CDR-H2 Residues 50-65 MIAADGFTDYASSVKG
    VH of SEQ ID
    NO.: 34
    A8 CDR-H3 Residues 98-106 EWHHGPVAY
    VH of SEQ ID
    NO.: 34
    B5 SEQ ID NO.: 35 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVSWVRQAPGKGLEWVSLIRGDGSTDYA
    SSLKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWHHGPVAYWGQGTLVTVSS
    B5 CDR-H1 Residues 31-35 NYGVS
    VH of SEQ ID
    NO.: 35
    B5 CDR-H2 Residues 50-65 LIRGDGSTDYASSLKG
    VH of SEQ ID
    NO.: 35
    B5 CDR-H3 Residues 98-106 EWHHGPVAY
    VH of SEQ ID
    NO.: 35
    rHC44 SEQ ID NO.: 36 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVSWVRQAPGKGLEWVSMIWADGSTHYA
    DTLKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC44 CDR-H1 Residues 31-35 NYGVS
    VH of SEQ ID
    NO.: 36
    rHC44 CDR-H2 Residues 50-65 MIWADGSTHYADTLKS
    VH of SEQ ID
    NO.: 36
    rHC44 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 36
    rHC22 SEQ ID NO.: 37 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTDYA
    DTVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC22 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 37
    rHC22 CDR-H2 Residues 50-65 MIWADGSTDYADTVKG
    VH of SEQ ID
    NO.: 37
    rHC22 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 37
    rHC81 SEQ ID NO.: 38 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    DSVKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPLAYWGQGTLVTVSS
    rHC81 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 38
    rHC81 CDR-H2 Residues 50-65 MIWADGSTHYADSVKS
    VH of SEQ ID
    NO.: 38
    rHC81 CDR-H3 Residues 98-106 EWQHGPLAY
    VH of SEQ ID
    NO.: 38
    rHC18 SEQ ID NO.: 39 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWSDGSTDYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC18 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 39
    rHC18 CDR-H2 Residues 50-65 MIWSDGSTDYASSVKG
    VH of SEQ ID
    NO.: 39
    rHC18 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 39
    rHC14 SEQ ID NO.: 40 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSLKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPAAYWGQGTLVTVSS
    rHC14 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 40
    rHC14 CDR-H2 Residues 50-65 MIWADGSTHYASSLKG
    VH of SEQ ID
    NO.: 40
    rHC14 CDR-H3 Residues 98-106 EWQHGPAAY
    VH of SEQ ID
    NO.: 40
    rHC3 SEQ ID NO.: 41 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVSWVRQAPGKGLEWVSMIWADGSTHYA
    SSLKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC3 CDR-H1 Residues 31-35 NYGVS
    VH of SEQ ID
    NO.: 41
    rHC3 CDR-H2 Residues 50-65 MIWADGSTHYASSLKG
    VH of SEQ ID
    NO.: 41
    rHC3 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 41
    rHC19 SEQ ID NO.: 42 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPAAYWGQGTLVTVSS
    rHC19 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 42
    rHC19 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 42
    rHC19 CDR-H3 Residues 98-106 EWQHGPAAY
    VH of SEQ ID
    NO.: 42
    rHC34 SEQ ID NO.: 43 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPSAYWGQGTLVTVSS
    rHC34 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 43
    rHC34 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 43
    rHC34 CDR-H3 Residues 98-106 EWQHGPSAY
    VH of SEQ ID
    NO.: 43
    rHC83 SEQ ID NO.: 44 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC83 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 44
    rHC83 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 44
    rHC83 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 44
    S4-19 SEQ ID NO.: 45 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVEWVRQAPGKGLEWVSGIWADGSTHYA
    DTVKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-19 CDR-H1 Residues 31-35 NYGVE
    VH of SEQ ID
    NO.: 45
    S4-19 CDR-H2 Residues 50-65 GIWADGSTHYADTVKS
    VH of SEQ ID
    NO.: 45
    S4-19 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 45
    S4-50 SEQ ID NO.: 46 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVEWVRQAPGKGLEWVSGIWADGSTHYA
    DTVKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVGYWGQGTLVTVSS
    S4-50 CDR-H1 Residues 31-35 NYGVE
    VH of SEQ ID
    NO.: 46
    S4-50 CDR-H2 Residues 50-65 GIWADGSTHYADTVKS
    VH of SEQ ID
    NO.: 46
    S4-50 CDR-H3 Residues 98-106 EWQHGPVGY
    VH of SEQ ID
    NO.: 46
    S4-63 SEQ ID NO.: 47 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVEWVRQAPGKGLEWVSGIWADGSTHYA
    DTVKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVGYWGQGTLVTVSS
    S4-63 CDR-H1 Residues 31-35 NYGVE
    VH of SEQ ID
    NO.: 47
    S4-63 CDR-H2 Residues 50-65 GIWADGSTHYADTVKS
    VH of SEQ ID
    NO.: 47
    S4-63 CDR-H3 Residues 98-106 EWQHGPVGY
    VH of SEQ ID
    NO.: 47
    S4-55 SEQ ID NO.: 48 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTDYA
    STVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVGYWGQGTLVTVSS
    S4-55 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 48
    S4-55 CDR-H2 Residues 50-65 MIWADGSTDYASTVKG
    VH of SEQ ID
    NO.: 48
    S4-55 CDR-H3 Residues 98-106 EWQHGPVGY
    VH of SEQ ID
    NO.: 48
    S4-6 SEQ ID NO.: 49 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-6 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 49
    S4-6 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 49
    S4-6 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 49
    S4-18 SEQ ID NO.: 50 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    DSVKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPLAYWGQGTLVTVSS
    S4-18 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 50
    S4-18 CDR-H2 Residues 50-65 MIWADGSTHYADSVKS
    VH of SEQ ID
    NO.: 50
    S4-18 CDR-H3 Residues 98-106 EWQHGPLAY
    VH of SEQ ID
    NO.: 50
    S4-31 SEQ ID NO.: 51 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVQWVRQAPGKGLEWVSGIGADGSTAYA
    SSLKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHSGLAYWGQGTLVTVSS
    S4-31 CDR-H1 Residues 31-35 NYGVQ
    VH of SEQ ID
    NO.: 51
    S4-31 CDR-H2 Residues 50-65 GIGADGSTAYASSLKG
    VH of SEQ ID
    NO.: 51
    S4-31 CDR-H3 Residues 98-106 EWQHSGLAY
    VH of SEQ ID
    NO.: 51
    S4-34 SEQ ID NO.: 52 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVSWVRQAPGKGLEWVSMIWADGSTHYA
    DTVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPLAYWGQGTLVTVSS
    S4-34 CDR-H1 Residues 31-35 NYGVS
    VH of SEQ ID
    NO.: 52
    S4-34 CDR-H2 Residues 50-65 MIWADGSTHYADTVKG
    VH of SEQ ID
    NO.: 52
    S4-34 CDR-H3 Residues 98-106 EWQHGPLAY
    VH of SEQ ID
    NO.: 52
    S4-74 SEQ ID NO.: 53 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    DTVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPLAYWGQGTLVTVSS
    S4-74 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 53
    S4-74 CDR-H2 Residues 50-65 MIWADGSTHYADTVKG
    VH of SEQ ID
    NO.: 53
    S4-74 CDR-H3 Residues 98-106 EWQHGPLAY
    VH of SEQ ID
    NO.: 53
    S4-12 SEQ ID NO.: 54 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-12 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 54
    S4-12 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 54
    S4-12 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 54
    S4-54 SEQ ID NO.: 55 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-54 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 55
    S4-54 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 55
    S4-54 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 55
    S4-17 SEQ ID NO.: 56 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-17 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 56
    S4-17 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 56
    S4-17 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 56
    S4-40 SEQ ID NO.: 57 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-40 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 57
    S4-40 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 57
    S4-40 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 57
    S4-24 SEQ ID NO.: 58 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-24 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 58
    S4-24 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 58
    S4-24 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 58
  • Table 7 provides VL sequences of IgG converted clones for Humanized anti-TNF MAK-195 antibodies as discussed in detail in Example 2.
  • TABLE 7
    Humanized anti-TNF MAK-195 Ab VL sequences of IgG
    converted clones
    Sequence
    Protein region 123456789012345678901234567890
    hMAK195 SEQ ID NO.: 59 DIQMTQSPSSLSASVGDRVTITCKASQAVS
    VL.1 SAVAWYQQKPGKAPKLLIYWASTRHTGVPS
    VL RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYSTPFTFGQGTKLEIKR
    hMAK195 CDR-L1 Residues 24-34 KASQAVSSAVA
    VL.1 of SEQ ID
    VL NO.: 59
    hMAK195 CDR-L2 Residues 50-56 WASTRHT
    VL.1 of SEQ ID
    VL NO.: 59
    hMAK195 CDR-L3 Residues 89-97 QQHYSTPFT
    VL.1 of SEQ ID
    VL NO.: 59
    S4-24 SEQ ID NO.: 60 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTLHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-24 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 60
    S4-24 CDR-L2 Residues 50-56 WASTLHT
    VL of SEQ ID
    NO.: 60
    S4-24 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 60
    S4-40 SEQ ID NO.: 61 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTRHSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFSFGQGTKLEIKR
    S4-40 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 61
    S4-40 CDR-L2 Residues 50-56 WASTRHS
    VL of SEQ ID
    NO.: 61
    S4-40 CDR-L3 Residues 89-97 QQHYRTPFS
    VL of SEQ ID
    NO.: 61
    S4-17 SEQ ID NO.: 62 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTRHSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-17 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 62
    S4-17 CDR-L2 Residues 50-56 WASTRHS
    VL of SEQ ID
    NO.: 62
    S4-17 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 62
    S4-54 SEQ ID NO.: 63 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASARHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYKTPFSFGQGTKLEIKR
    S4-54 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 63
    S4-54 CDR-L2 Residues 50-56 WASARHT
    VL of SEQ ID
    NO.: 63
    S4-54 CDR-L3 Residues 89-97 QQHYKTPFS
    VL of SEQ ID
    NO.: 63
    S4-12 SEQ ID NO.: 64 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASARHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYKTPFTFGQGTKLEIKR
    S4-12 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 64
    S4-12 CDR-L2 Residues 50-56 WASARHT
    VL of SEQ ID
    NO.: 64
    S4-12 CDR-L3 Residues 89-97 QQHYKTPFT
    VL of SEQ ID
    NO.: 64
    S4-74 SEQ ID NO.: 65 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASARHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-74 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 65
    S4-74 CDR-L2 Residues 50-56 WASARHT
    VL of SEQ ID
    NO.: 65
    S4-74 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 65
    S4-34 SEQ ID NO.: 66 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTRHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-34 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 66
    S4-34 CDR-L2 Residues 50-56 WASTRHT
    VL of SEQ ID
    NO.: 66
    S4-34 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 66
    S4-31 SEQ ID NO.: 67 DIQMTQSPSSLSASVGDRVTITCRASQGVS
    VL SALAWYQQKPGKAPKLLIYWASALHSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYSAPFTFGQGTKLEIKR
    S4-31 CDR-L1 Residues 24-34 RASQGVSSALA
    VL of SEQ ID
    NO.: 67
    S4-31 CDR-L2 Residues 50-56 WASALHS
    VL of SEQ ID
    NO.: 67
    S4-31 CDR-L3 Residues 89-97 QQHYSAPFT
    VL of SEQ ID
    NO.: 67
    S4-18 SEQ ID NO.: 68 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTLHSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYSTPFTFGQGTKLEIKR
    S4-18 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 68
    S4-18 CDR-L2 Residues 50-56 WASTLHS
    VL of SEQ ID
    NO.: 68
    S4-18 CDR-L3 Residues 89-97 QQHYSTPFT
    VL of SEQ ID
    NO.: 68
    S4-6 SEQ ID NO.: 69 DIQMTQSPSSLSASVGDRVTITCKASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTRHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYSTPFTFGQGTKLEIKR
    S4-6 CDR-L1 Residues 24-34 KASQLVSSAVA
    VL of SEQ ID
    NO.: 69
    S4-6 CDR-L2 Residues 50-56 WASTRHT
    VL of SEQ ID
    NO.: 69
    S4-6 CDR-L3 Residues 89-97 QQHYSTPFT
    VL of SEQ ID
    NO.: 69
    S4-55 SEQ ID NO.: 70 DIQMTQSPSSLSASVGDRVTITCKASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTLHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-55 CDR-L1 Residues 24-34 KASQLVSSAVA
    VL of SEQ ID
    NO.: 70
    S4-55 CDR-L2 Residues 50-56 WASTLHT
    VL of SEQ ID
    NO.: 70
    S4-55 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 70
    S4-63 SEQ ID NO.: 71 DIQMTQSPSSLSASVGDRVTITCKASQKVS
    VL SALAWYQQKPGKAPKLLIYWASALHSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRPPFTFGQGTKLEIKR
    S4-63 CDR-L1 Residues 24-34 KASQKVSSALA
    VL of SEQ ID
    NO.: 71
    S4-63 CDR-L2 Residues 50-56 WASALHS
    VL of SEQ ID
    NO.: 71
    S4-63 CDR-L3 Residues 89-97 QQHYRPPFT
    VL of SEQ ID
    NO.: 71
    S4-50 SEQ ID NO.: 72 DIQMTQSPSSLSASVGDRVTITCKASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASALHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYSSPYTFGQGTKLEIKR
    S4-50 CDR-L1 Residues 24-34 KASQLVSSAVA
    VL of SEQ ID
    NO.: 72
    S4-50 CDR-L2 Residues 50-56 WASALHT
    VL of SEQ ID
    NO.: 72
    S4-50 CDR-L3 Residues 89-97 QQHYSSPYT
    VL of SEQ ID
    NO.: 72
    S4-19 SEQ ID NO.: 73 DIQMTQSPSSLSASVGDRVTITCKASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTLHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-19 CDR-L1 Residues 24-34 KASQLVSSAVA
    VL of SEQ ID
    NO.: 73
    S4-19 CDR-L2 Residues 50-56 WASTLHT
    VL of SEQ ID
    NO.: 73
    S4-19 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 73
  • C. Individual hMAK-199 Sequences from Converted Clones
  • Table 8 provides VH sequences of humanized anti-TNF MAK-199 converted clones as discussed in detail in Example 3.
  • TABLE 8
    Humanized Anti-TNF MAK-199 Ab VH sequences of IgG
    converted clones
    Sequence
    Protein region 123456789012345678901234567890
    J662M2S3 SEQ ID NO.: 74 EVQLVQSGAEVKKPGASVKVSCKASGYTFA
    #10 VH NYGIIWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYYCARKLFTTMDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    10 VH of SEQ ID
    NO.: 74
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    10 VH of SEQ ID
    NO.: 74
    J662M2S3# CDR-H3 Residues 99-112 RASQDISQYLN
    10 VH of SEQ ID
    NO.: 74
    J662M2S3# SEQ ID NO.: 75 EVQLVQSGAEVKKPGASVKVSCKASGYTFN
    13 VH NYGIIWVRQAPGQGLEWMGWINTYTGKPTY
    AQKLQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYFCARKLFNTVDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    13 VH of SEQ ID
    NO.: 75
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKLQG
    13 VH of SEQ ID
    NO.: 75
    J662M2S3# CDR-H3 Residues 99-112 KLFNTVDVTDNAMD
    13 VH of SEQ ID
    NO.: 75
    J662M2S3# SEQ ID NO.: 76 EVQLVQSGAEVKKPGASVKVSCKASGYTFN
    15 VH NYGIIWVRQAPGQGLEWMGWINTYTGVPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYYCARKLFNTVDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    15 VH of SEQ ID
    NO.: 76
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGVPTYAQKFQG
    15 VH of SEQ ID
    NO.: 76
    J662M2S3# CDR-H3 Residues 99-112 KLFNTVDVTDNAMD
    15 VH of SEQ ID
    NO.: 76
    J662M2S3# SEQ ID NO.: 77 EVQLVQSGAEVKKPGASVKVSCKASGYTFN
    16 VH NYGIIWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYYCARKLFNTVAVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    16 VH of SEQ ID
    NO.: 77
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    16 VH of SEQ ID
    NO.: 77
    J662M2S3# CDR-H3 Residues 99-112 KLFNTVAVTDNAMD
    16 VH of SEQ ID
    NO.: 77
    J662M2S3# SEQ ID NO.: 78 EVQLVQSGAEVKKPGASVKVSCKASGYTFR
    21 VH NYGIIWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYFCARKLFTTVDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    21 VH of SEQ ID
    NO.: 78
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    21 VH of SEQ ID
    NO.: 78
    J662M2S3# CDR-H3 Residues 99-112 KLFTTVDVTDNAMD
    21 VH of SEQ ID
    NO.: 78
    J662M2S3# SEQ ID NO.: 79 EVQLVQSGAEVKKPGASVKVSCKASGYTFN
    34 VH NYGINWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYFCARKFRNTVAVTDYAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGIN
    34 VH of SEQ ID
    NO.: 79
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    34 VH of SEQ ID
    NO.: 79
    J662M2S3# CDR-H3 Residues 99-112 KFRNTVAVTDYAMD
    34 VH of SEQ ID
    NO.: 79
    J662M2S3# SEQ ID NO.: 80 EVQLVQSGAEVKKPGASVKVSCKASGYTFR
    36 VH NYGITWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYFCARKLFTTMDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGIT
    36 VH of SEQ ID
    NO.: 80
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    36 VH of SEQ ID
    NO.: 80
    J662M2S3# CDR-H3 Residues 99-112 KLFTTMDVTDNAMD
    36 VH of SEQ ID
    NO.: 80
    J662M2S3# SEQ ID NO.: 81 EVQLVQSGAEVKKPGASVKVSCKASGYTFA
    45 VH NYGIIWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYYCARKLFTTMDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    45 VH of SEQ ID
    NO.: 81
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    45 VH of SEQ ID
    NO.: 81
    J662M2S3# CDR-H3 Residues 99-112 KLFTTMDVTDNAMD
    45 VH of SEQ ID
    NO.: 81
    J662M2S3# SEQ ID NO.: 82 EVQLVQSGAEVKKPGASVKVSCKASGYTFS
    58 VH NYGINWVRQAPGQGLEWMGWINTYTGQPSY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYYCARKLFKTEAVTDYAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGIN
    58 VH of SEQ ID
    NO.: 82
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGQPSYAQKFQG
    58 VH of SEQ ID
    NO.: 82
    J662M2S3# CDR-H3 Residues 99-112 KLFKTEAVTDYAMD
    58 VH of SEQ ID
    NO.: 82
    J662M2S3# SEQ ID NO.: 83 EVQLVQSGAEVKKPGASVKVSCKASGYTFN
    72 VH NYGIIWVRQAPGQGLEWMGWINTYSGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYFCARKLFTTMDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    72 VH of SEQ ID
    NO.: 83
    J662M2S3# CDR-H2 Residues 50-66 WINTYSGKPTYAQKFQG
    72 VH of SEQ ID
    NO.: 83
    J662M2S3# CDR-H3 Residues 99-112 KLFTTMDVTDNAMD
    72 VH of SEQ ID
    NO.: 83
  • Table 9 provides VL sequences of humanized anti-TNF MAK-199 converted clones as discussed in detail in Example 3.
  • TABLE 9
    Humanized Anti-TNF MAK-199 Ab VL sequences of IgG
    converted clones
    Sequence
    Protein region 123456789012345678901234567890
    J662M2S3# SEQ ID DIQMTQSPSSLSASVGDRVTITCRASQDIS
    10 VL NO.: 84 QYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
    GNTWPPTFGQGTKLEIK
    J662M2S3#10 CDR-L1 Residues 24-34 RASQDISQYLN
    VL of SEQ ID
    NO.: 84
    J662M2S3#10 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 84
    J662M2S3#10 CDR-L3 Residues 89-97 QQGNTWPPT
    VL of SEQ
    ID NO.: 84
    J662M2S3#13 SEQ ID DIQMTQSPSSLSASVGDRVTITCRASQDIS
    VL NO.: 85 NYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDYTLTISSLQPEDFATYFCQQ
    GNSWPPTFGQGTKLEIK
    J662M2S3#13 CDR-L1 Residues 24-34 RASQDISNYLN
    VL of SEQ ID
    NO.: 85
    J662M2S3#13 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 85
    J662M2S3#13 CDR-L3 Residues 89-97 QQGNSWPPT
    VL of SEQ
    ID NO.: 85
    J662M2S3#15 SEQ ID DIQMTQSPSSLSASVGDRVTITCRASQDIY
    VL NO.: 86 NYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDYTLTISSLQPEDFATYFCQQ
    GNTQPPTFGQGTKLEIK
    J662M2S3#15 CDR-L1 Residues 24-34 RASQDIYNYLN
    VL of SEQ ID
    NO.: 86
    J662M2S3#15 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 86
    J662M2S3#15 CDR-L3 Residues 89-97 QQGNTQPPT
    VL of SEQ
    ID NO.: 86
    J662M2S3#16 SEQ ID DIQMTQSPSSLSASVGDRVTITCRASQDIE
    VL NO.: 87 NYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
    GNTQPPTFGQGTKLEIK
    J662M2S3#16 CDR-L1 Residues 24-34 RASQDIENYLN
    VL of SEQ ID
    NO.: 87
    J662M2S3#16 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 87
    J662M2S3#16 CDR-L3 Residues 89-97 QQGNTQPPT
    VL of SEQ
    ID NO.: 87
    J662M2S3#21 SEQ ID DIQMTQSPSSLSASVGDRVTITCRASQDIS
    VL NO.: 88 NYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDYTLTISSLQPEDFATYFCQQ
    GNTWPPTFGQGTKLEIK
    J662M2S3#21 CDR-L1 Residues 24-34 RASQDISNYLN
    VL of SEQ ID
    NO.: 88
    J662M2S3#21 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 88
    J662M2S3#21 CDR-L3 Residues 89-97 QQGNTWPPT
    VL of SEQ
    ID NO.: 88
    J662M2S3#34 SEQ ID DIQMTQSPSSLSASVGDRVTITCRASQDIY
    VL NO.: 89 DVLNWYQQKPGKAPKLLIYYASRLQSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    GITLPPTFGQGTKLEIK
    J662M2S3#34 CDR-L1 Residues 24-34 RASQDIYDVLN
    VL of SEQ ID
    NO.: 89
    J662M2S3#34 CDR-L2 Residues 50-56 YASRLQS
    VL of SEQ ID
    NO.: 89
    J662M2S3#34 CDR-L3 Residues 89-97 QQGITLPPT
    VL of SEQ
    ID NO.: 89
    J662M2S3#36 SEQ ID DIQMTQSPSSLSASVGDRVTITCRASQDIS
    VL NO.: 90 NYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDYTLTISSLQPEDFATYFCQQ
    GNTWPPTFGQGTKLEIK
    J662M2S3#36 CDR-L1 Residues 24-34 RASQDISNYLN
    VL of SEQ ID
    NO.: 90
    J662M2S3#36 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 90
    J662M2S3#36 CDR-L3 Residues 89-97 QQGNTWPPT
    VL of SEQ
    ID NO.: 90
    J662M2S3#45 SEQ ID DIQMTQSPSSLSASVGDRVTITCRASQDIS
    VL NO.: 91 QYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
    GNTWPPTFGQGTKLEIK
    J662M2S3#45 CDR-L1 Residues 24-34 RASQDISQYLN
    VL of SEQ ID
    NO.: 91
    J662M2S3#45 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 91
    J662M2S3#45 CDR-L3 Residues 89-97 QQGNTWPPT
    VL of SEQ
    ID NO.: 91
    J662M2S3#58 SEQ ID DIQMTQSPSSLSASVGDRVTITCRASQNIY
    VL NO.: 92 NVLNWYQQKPGKAPKLLIYYASRLQSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
    GNTMPPTFGQGTKLEIK
    J662M2S3#58 CDR-L1 Residues 24-34 RASQNIYNVLN
    VL of SEQ ID
    NO.: 92
    J662M2S3#58 CDR-L2 Residues 50-56 YASRLQS
    VL of SEQ ID
    NO.: 92
    J662M2S3#58 CDR-L3 Residues 89-97 QQGNTMPPT
    VL of SEQ
    ID NO.: 92
    J662M2S3#72 SEQ ID DIQMTQSPSSLSASVGDRVTITCRASQDIS
    VL NO.: 93 NFLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDYTLTISSLQPEDFATYFCQQ
    GNTQPPTFGQGTKLEIK
    J662M2S3#72 CDR-L1 Residues 24-34 RASQDISNFLN
    VL of SEQ ID
    NO.: 93
    J662M2S3#72 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 93
    J662M2S3#72 CDR-L3 Residues 89-97 QQGNTQPPT
    VL of SEQ
    ID NO.: 93
  • In an embodiment, the antigen binding domain comprises the VH region chosen from any one of SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34-58, 74-83, 94-266, 478-486, 496-675, 738-762, 778-956, 1053-1062, 1073, 1075, and 1077, or one, two, or three CDRs therefrom. In another embodiment, the antigen binding domain comprises the VL region chosen from any one of SEQ ID NOs: 23, 25, 27, 29, 31, 33, 59-73, 84-93, 267-477, 487-495, 676-737, 763-777, 957-1052, 1063-1072, 1074, 1076, and 1078, or one, two, or three CDRs therefrom. In a particular embodiment, the antigen binding domain comprises a VH region and a VL region, for example, wherein the VH region comprises SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34-58, 74-83, 94-266, 478-486, 496-675, 738-762, 778-956, 1053-1062, 1073, 1075, and 1077, or one, two, or three CDRs therefrom, and the VL region comprises SEQ ID NOs: 23, 25, 27, 29, 31, 33, 59-73, 84-93, 267-477, 487-495, 676-737, 763-777, 957-1052, 1063-1072, 1074, 1076, and 1078, or one, two, or three CDRs therefrom.
  • In an embodiment where the VH and/or the VL CDR sequences are provided above, the human acceptor framework comprises at least one amino acid sequence selected from: SEQ ID NOs: 6-21. In a particular embodiment, the human acceptor framework comprises an amino acid sequence selected from: SEQ 1N NOs: 9, 10, 11, 12, 15, 16, 17, and 21. In another embodiment, the human acceptor framework comprises at least one framework region amino acid substitution, wherein the amino acid sequence of the framework is at least 65% identical to the sequence of the human acceptor framework and comprises at least 70 amino acid residues identical to the human acceptor framework. In another embodiment, the human acceptor framework comprises at least one framework region amino acid substitution at a key residue. The key residue selected from: a residue adjacent to a CDR; a glycosylation site residue; a rare residue; a residue capable of interacting with human TNF-α; a residue capable of interacting with a CDR; a canonical residue; a contact residue between heavy chain variable region and light chain variable region; a residue within a Vernier zone; and a residue in a region that overlaps between a Chothia-defined variable heavy chain CDR1 and a Kabat-defined first heavy chain framework. In an embodiment, the key residue is selected from: H1, H12, H24, H27, H29, H37, H48, H49, H67, H71, H73, H76, H78, L13, L43, L58, L70, and L80. In an embodiment, the VH mutation is selected from: Q1E, I12V, A24V, G27F, I29L, V29F F29L 137V, I48L, V48L, S49G, V67L, F67L, V71K, R71K, T73N, N76S, L78I, and F78I. In another embodiment, the VL mutation is selected from: V13L, A435, I58V, E70D, and S80P. In an embodiment, the binding protein comprises two variable domains, wherein the two variable domains have amino acid sequences selected from: SEQ ID NOS: 22 and 23; 23 and 24; 24 and 25; 26 and 27; 28 and 29; 30 and 31; or 32 and 33.
    • III. Production of Binding Proteins and Binding Protein-Producing Cell Lines
  • In an embodiment, TNF-α binding proteins disclosed herein exhibit a high capacity to reduce or to neutralize TNF-α activity, e.g., as assessed by any one of several in vitro and in vivo assays known in the art. Alternatively, TNF-α binding proteins disclosed herein, also exhibit a high capacity to increase or agonize TNF-α activity.
  • In particular embodiments, the isolated binding protein, or antigen-binding portion thereof, binds human TNF-α, wherein the binding protein, or antigen-binding portion thereof, dissociates from human TNF-α with a koff rate constant of about 0.1 s−1 or less, as determined by surface plasmon resonance, such as 1×10−2 s−1 or less, 1×10−3 s−1 or less, 1×10−4 s−1 or less, 1×10−5 s−1 or less and 1×10−6 s−1 or less; or which inhibits human TNF-α activity with an IC50 of about 1×10−6 M or less, such as 1×10−7M or less, 1×10−8M or less, 1×10−9M or less, 1×10−10 M or less and 1×1011 M or less. In certain embodiments, the binding protein comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. In an embodiment, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the binding protein can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region. In another embodiment, the binding protein comprises a kappa light chain constant region. Alternatively, the binding protein portion can be, for example, a Fab fragment or a single chain Fv fragment.
  • Replacements of amino acid residues in the Fc portion to alter binding protein effector function are known in the art (See U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of a binding protein mediates several important effector functions, e.g., cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRs and complement C1q, respectively. Neonatal Fc receptors (FcRn) are the critical components determining the circulating half-life of antibodies. In still another embodiment at least one amino acid residue is replaced in the constant region of the binding protein, for example the Fc region of the binding protein, such that effector functions of the binding protein are altered.
  • One embodiment provides a labeled binding protein wherein an antibody or antibody portion disclosed herein is derivatized or linked to another functional molecule (e.g., another peptide or protein). For example, a labeled binding protein disclosed herein can be derived by functionally linking an antibody or antibody portion disclosed herein (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • Useful detectable agents with which an antibody or antibody portion disclosed herein may be derivatized include fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like. An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. An antibody may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
  • Another embodiment of the disclosure provides a crystallized binding protein. In an embodiment, provided are crystals of whole TNF-α binding proteins and fragments thereof as disclosed herein, and formulations and compositions comprising such crystals. In one embodiment the crystallized binding protein has a greater half-life in vivo than the soluble counterpart of the binding protein. In another embodiment the binding protein retains biological activity after crystallization.
  • Crystallized binding protein disclosed herein may be produced according methods known in the art and as disclosed in PCT Publication WO 02/72636.
  • Another embodiment of the disclosure provides a glycosylated binding protein wherein the binding protein or antigen-binding portion thereof comprises one or more carbohydrate residues. Nascent in vivo protein production may undergo further processing, known as post-translational modification. In particular, sugar (glycosyl) residues may be added enzymatically, a process known as glycosylation. The resulting proteins bearing covalently linked oligosaccharide side chains are known as glycosylated proteins or glycoproteins. Protein glycosylation depends on the amino acid sequence of the protein of interest, as well as the host cell in which the protein is expressed. Different organisms may produce different glycosylation enzymes (e.g., glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available. Due to such factors, protein glycosylation pattern, and composition of glycosyl residues, may differ depending on the host system in which the particular protein is expressed. Glycosyl residues useful in the disclosure may include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. In an embodiment, the glycosylated binding protein comprises glycosyl residues such that the glycosylation pattern is human.
  • It is known to those skilled in the art that differing protein glycosylation may result in differing protein characteristics. For instance, the efficacy of a therapeutic protein produced in a microorganism host, such as yeast, and glycosylated utilizing the yeast endogenous pathway may be reduced compared to that of the same protein expressed in a mammalian cell, such as a CHO cell line. Such glycoproteins may also be immunogenic in humans and show reduced half-life in vivo after administration. Specific receptors in humans and other animals may recognize specific glycosyl residues and promote the rapid clearance of the protein from the bloodstream. Other adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. Accordingly, a practitioner may prefer a therapeutic protein with a specific composition and pattern of glycosylation, for example glycosylation composition and pattern identical, or at least similar, to that produced in human cells or in the species-specific cells of the intended subject animal.
  • Expressing glycosylated proteins different from that of a host cell may be achieved by genetically modifying the host cell to express heterologous glycosylation enzymes. Using techniques known in the art a practitioner may generate antibodies or antigen-binding portions thereof exhibiting human protein glycosylation. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylation enzymes such that glycosylated proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation identical to that of animal cells, especially human cells (U.S. Pat. Nos. 7,449,308 and 7,029,872).
  • Further, it will be appreciated by one skilled in the art that a protein of interest may be expressed using a library of host cells genetically engineered to express various glycosylation enzymes, such that member host cells of the library produce the protein of interest with variant glycosylation patterns. A practitioner may then select and isolate the protein of interest with particular novel glycosylation patterns. In an embodiment, the protein having a particularly selected novel glycosylation pattern exhibits improved or altered biological properties.
    • IV. Uses of TNF-α Binding Proteins
  • Given their ability to bind to human TNF-α, e.g., the human TNF-α binding proteins, or portions thereof, disclosed herein can be used to detect TNF-α (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. A method for detecting TNF-α in a biological sample is provided comprising contacting a biological sample with a binding protein, or binding protein portion, disclosed herein and detecting either the binding protein (or binding protein portion) bound to TNF-α or unbound binding protein (or binding protein portion), to thereby detect TNF-α in the biological sample. The binding protein is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm.
  • Alternative to labeling the binding protein, human TNF-α can be assayed in biological fluids by a competition immunoassay utilizing rhTNF-α standards labeled with a detectable substance and an unlabeled human TNF-α binding protein. In this assay, the biological sample, the labeled rhTNF-α standards and the human TNF-α binding protein are combined and the amount of labeled rhTNF-α standard bound to the unlabeled binding protein is determined. The amount of human TNF-α in the biological sample is inversely proportional to the amount of labeled rhTNF-α standard bound to the TNF-α binding protein. Similarly, human TNF-α can also be assayed in biological fluids by a competition immunoassay utilizing rhTNF-α standards labeled with a detectable substance and an unlabeled human TNF-α binding protein.
  • In an embodiment, the binding proteins and binding protein portions disclosed herein are capable of neutralizing TNF-α activity, e.g., human TNF-α activity, both in vitro and in vivo. In another embodiment, the binding proteins and binding protein portions disclosed herein are capable of increasing or agonizing human TNF-α activity, e.g., human TNF-α activity. Accordingly, such binding proteins and binding protein portions disclosed herein can be used to inhibit or increase hTNF-α activity, e.g., in a cell culture containing hTNF-α, in human subjects or in other mammalian subjects having TNF-α with which a binding protein disclosed herein cross-reacts. In one embodiment, a method for inhibiting or increasing hTNF-α activity is provided comprising contacting hTNF-α with a binding protein or binding protein portion disclosed herein such that hTNF-α activity is inhibited or increased. For example, in a cell culture containing, or suspected of containing hTNF-α, a binding protein or binding protein portion disclosed herein can be added to the culture medium to inhibit or increase hTNF-α activity in the culture.
  • In another embodiment, a method is provided for reducing or increasing hTNF-α activity in a subject, advantageously from a subject suffering from a disease or disorder in which TNF-α activity is detrimental or, alternatively, beneficial. Methods for reducing or increasing TNF-α activity in a subject suffering from such a disease or disorder is provided, which method comprises administering to the subject a binding protein or binding protein portion disclosed herein such that TNF-α activity in the subject is reduced or increased. In a particular embodiment, the TNF-α is human TNF-α, and the subject is a human subject. Alternatively, the subject can be a mammal expressing a TNF-α to which a binding protein provided is capable of binding. Still further the subject can be a mammal into which TNF-α has been introduced (e.g., by administration of TNF-α or by expression of a TNF-α transgene). A binding protein disclosed herein can be administered to a human subject for therapeutic purposes. Moreover, a binding protein disclosed herein can be administered to a non-human mammal expressing a TNF-α with which the binding protein is capable of binding for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of binding proteins disclosed herein (e.g., testing of dosages and time courses of administration).
  • The term “a disorder in which TNF-α activity is detrimental” includes diseases and other disorders in which the presence of TNF-α activity in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which TNF-α activity is detrimental is a disorder in which reduction of TNF-α activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of TNF-α in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of TNF-α in serum, plasma, synovial fluid, etc. of the subject), which can be detected, for example, using an anti-TNF-α antibody as described above. Non-limiting examples of disorders that can be treated with the binding proteins disclosed herein include those disorders discussed in the section below pertaining to pharmaceutical compositions of the antibodies disclosed herein.
  • Alternatively, the term “a disorder in which TNF-α activity is beneficial” include diseases and other disorders in which the presence of TNF-α activity in a subject suffering from the disorder has been shown to be or is suspected of being either beneficial for treating the pathophysiology of the disorder or a factor that contributes to a treatment of the disorder. Accordingly, a disorder in which TNF-α activity is beneficial is a disorder in which an increase of TNF-α activity is expected to alleviate the symptoms and/or progression of the disorder. Non-limiting examples of disorders that can be treated with the antibodies disclosed herein include those disorders discussed in the section below pertaining to pharmaceutical compositions of the antibodies disclosed herein.
    • V. Pharmaceutical Compositions
  • Pharmaceutical compositions are also provided comprising a binding protein, or antigen-binding portion thereof, disclosed herein and a pharmaceutically acceptable carrier. The pharmaceutical compositions comprising binding protein disclosed herein are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder, in preventing, treating, managing, or ameliorating of a disorder or one or more symptoms thereof, and/or in research. In a specific embodiment, a composition comprises one or more binding proteins disclosed herein. In another embodiment, the pharmaceutical composition comprises one or more binding proteins disclosed herein and one or more prophylactic or therapeutic agents other than binding proteins disclosed herein for treating a disorder in which TNF-α activity is detrimental. In a particular embodiment, the prophylactic or therapeutic agents known to be useful for or having been or currently being used in the prevention, treatment, management, or amelioration of a disorder or one or more symptoms thereof. In accordance with these embodiments, the composition may further comprise of a carrier, diluent or excipient.
  • The binding proteins and binding protein-portions disclosed herein can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises a binding protein or binding protein portion disclosed herein and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition, may be included. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding protein or binding protein portion.
  • Various delivery systems are known and can be used to administer one or more binding proteins disclosed herein or the combination of one or more binding proteins disclosed herein and a prophylactic agent or therapeutic agent useful for preventing, managing, treating, or ameliorating a disorder or one or more symptoms thereof, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the binding protein or binding protein fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu (1987) J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of administering a prophylactic or therapeutic agent disclosed herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes). In addition, pulmonary administration can be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. In one embodiment, a binding protein disclosed herein, combination therapy, or a composition disclosed herein is administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.). In a specific embodiment, prophylactic or therapeutic agents disclosed herein are administered intramuscularly, intravenously, intratumorally, orally, intranasally, pulmonary, or subcutaneously. The prophylactic or therapeutic agents may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents Administration can be systemic or local.
  • In a specific embodiment, it may be desirable to administer the prophylactic or therapeutic agents disclosed herein locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous or non-porous material, including membranes and matrices, such as sialastic membranes, polymers, fibrous matrices (e.g., Tissuel®), or collagen matrices. In one embodiment, an effective amount of one or more binding proteins disclosed herein antagonists is administered locally to the affected area to a subject to prevent, treat, manage, and/or ameliorate a disorder or a symptom thereof. In another embodiment, an effective amount of one or more binding proteins disclosed herein is administered locally to the affected area in combination with an effective amount of one or more therapies (e.g., one or more prophylactic or therapeutic agents) other than an antibody disclosed herein of a subject to prevent, treat, manage, and/or ameliorate a disorder or one or more symptoms thereof.
  • In a specific embodiment, where the composition disclosed herein is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, DuPont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see, e.g., Joliot et al. (1991) Proc. Natl. Acad. Sci. USA 88:1864-1868). Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.
  • The method disclosed herein may comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
  • The methods disclosed herein may additionally comprise of administration of compositions formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
  • The methods disclosed herein encompass administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the mode of administration is infusion, composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • In particular, it is also provided that one or more of the prophylactic or therapeutic agents, or pharmaceutical compositions disclosed herein is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent. In one embodiment, one or more of the prophylactic or therapeutic agents, or pharmaceutical compositions disclosed herein is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions disclosed herein is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. The lyophilized prophylactic or therapeutic agents or pharmaceutical compositions disclosed herein should be stored at between 2° C. and 8° C. in its original container and the prophylactic or therapeutic agents, or pharmaceutical compositions disclosed herein should be administered within 1 week, within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In an alternative embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions disclosed herein is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent. In an embodiment, the liquid form of the administered composition is supplied in a hermetically sealed container at least 0.25 mg/ml, at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid form should be stored at between 2° C. and 8° C. in its original container.
  • The binding proteins and binding protein-portions disclosed herein can be incorporated into a pharmaceutical composition suitable for parenteral administration. In an embodiment, the binding protein or binding protein-portions will be prepared as an injectable solution containing 0.1-250 mg/ml binding protein. The injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampoule or pre-filled syringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents can be included for a lyophilized dosage form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can be used in both liquid and lyophilized dosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking agents include glycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants.
  • Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., binding protein or binding protein portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including, in the composition, an agent that delays absorption, for example, monostearate salts and gelatin.
  • As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, a binding protein or binding protein portion disclosed herein is coformulated with and/or coadministered with one or more additional therapeutic agents that are useful for treating disorders in which TNF-α activity is detrimental. For example, an anti-hTNF-α antibody or antibody portion disclosed herein may be coformulated and/or coadministered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules). Furthermore, one or more binding proteins disclosed herein may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
  • In certain embodiments, a binding protein to TNF-α or fragment thereof is linked to a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, the Fc domain, polyethylene glycol, and dextran. Such vehicles are described, e.g., in U.S. Pat. No. 6,660,843.
  • In a specific embodiment, nucleic acid sequences comprising nucleotide sequences encoding a binding protein disclosed herein or another prophylactic or therapeutic agent disclosed herein are administered to treat, prevent, manage, or ameliorate a disorder or one or more symptoms thereof by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the disclosure, the nucleic acids produce their encoded binding protein or prophylactic or therapeutic agent disclosed herein that mediates a prophylactic or therapeutic effect.
  • Any of the methods for gene therapy available in the art can be used according to the present disclosure.
  • TNF-α plays a critical role in the pathology associated with a variety of diseases involving immune and inflammatory elements, such as autoimmune diseases, particularly those associated with inflammation, including Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, systemic lupus erythematosus, and ankylosing spondylitis. Therefore, the binding proteins herein may be used to treat these disorders. In another embodiment, the disorder is a respiratory disorder; asthma; allergic and nonallergic asthma; asthma due to infection; asthma due to infection with respiratory syncytial virus (RSV); chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation; eosinophilia; fibrosis and excess mucus production; cystic fibrosis; pulmonary fibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema; allergic rhinitis; allergic enterogastritis; an inflammatory and/or autoimmune condition of the skin; an inflammatory and/or autoimmune condition of gastrointestinal organs; inflammatory bowel diseases (IBD); ulcerative colitis; an inflammatory and/or autoimmune condition of the liver; liver cirrhosis; liver fibrosis; liver fibrosis caused by hepatitis B and/or C virus; scleroderma; tumors or cancers; hepatocellular carcinoma; glioblastoma; lymphoma; Hodgkin's lymphoma; a viral infection; a bacterial infection; a parasitic infection; HTLV-1 infection; suppression of expression of protective type 1 immune responses, suppression of expression of a protective type 1 immune response during vaccination, neurodegenerative diseases, neuronal regeneration, and spinal cord injury.
  • It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods disclosed herein may be made using suitable equivalents without departing from the scope of the invention or the embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting of the invention.
    • EXAMPLES Example 1 Identification of Fully Human Antibodies to TNF by In Vitro Display Systems 1.1: Antibody Selections
  • Fully human anti-human TNF monoclonal antibodies were isolated by in vitro display technologies from human antibody libraries by their ability to bind recombinant human TNF proteins. The amino acid sequences of the variable heavy (VH) and variable light (VL) chains were determined from DNA sequencing and listed in Table 10.
  • TABLE 10
    Individual clones sequences
    Sequence
    Protein region SEQ ID NO: 123456789012345678901234567890
    AE11-1 VH 22 EVQLVQSGAEVKKPGASVKVSCKASGYTFT
    SYDVN WVRQATGQGLEWMG WMNPNSGNTGY
    AQKFQG RVTITADESTSTAYMELSSLRSED
    TAVYYCAI FDSDYMDV WGKGTLVTVSS
    AE11-1 VH CDR- Residues 31-35 SYDVN
    H1 of SEQ ID
    NO.: 22
    AE11-1 VH CDR- Residues 50-66 WMNPNSGNTGYAQKFQG
    H2 of SEQ ID
    NO.: 22
    AE11-1 VH CDR- Residues 99-106 FDSDYMDV
    H3 of SEQ ID
    NO.: 22
    AE11-1 VL 23 SYELTQPPSVSLSPGQTARITC SGDALPKQ
    YAY WYQQKPGQAPVLVIY KDTERPS GIPER
    FSGSSSGTTVTLTISGAQAEDEADYYCQ SA
    DSSGTSWV FGGGTKLTVL
    AE11-1 VL CDR- Residues 23-33 SGDALPKQYAY
    L1 of SEQ ID
    NO.: 23
    AE11-1 VL CDR- Residues 49-55 KDTERPS
    L2 of SEQ ID
    NO.: 23
    AE11-1 VL CDR- Residues 89-98 SADSSGTSWV
    L3 of SEQ ID
    NO.: 23
    AE11-5 VH 24 EVQLVQSGAEVKKPGSSAKVSCKASGGTFS
    SYAIS WVRQAPGQGLEWMG GIIPILGTANY
    AQKFLG RVTITADESTSTVYMELSSLRSED
    TAVYYCAR GLYYDPTRADY WGQGTLVTVSS
    AE11-5 VH CDR- Residues 31-35 SYAIS
    H1 of SEQ ID
    NO.: 24
    AE11-5 VH CDR- Residues 50-66 GIIPILGTANYAQKFLG
    H2 of SEQ ID
    NO.: 24
    AE11-5 VH CDR- Residues 99-109 GLYYDPTRADY
    H3 of SEQ ID
    NO.: 24
    AE11-5 VL 25 DIVMTQSPDFHSVTPKEKVTITC RASQSIG
    SSLH WYQQKPDQSPKLLIR HASQSIS GVPS
    RFSGSGSGTDFTLTIHSLEAEDAATYYC HQ
    SSSSPPPT FGQGTQVEIK
    AE11-5 VL CDR- Residues 24-34 RASQSIGSSLH
    L1 of SEQ ID
    NO.: 25
    AE11-5 VL CDR- Residues 50-56 HASQSIS
    L2 of SEQ ID
    NO.: 25
    AE11-5 VL CDR- Residues 89-98 HQSSSSPPPT
    L3 of SEQ ID
    NO.: 25
    TNF-JK1 VH 26 EVQLVESGGGLVQPGGSLRLSCATSGFTFN
    NYWMS WVRQAPGKGLEWVA NINHDESEKYY
    VDSAKG RFTISRDNAEKSLFLQMNSLRAED
    TAVYYCAR IIRGRVGFDYYNYAMDV WGQGT
    LVTVSS
    TNF-JK1 VH CDR- Residues 31-35 NYWMS
    H1 of SEQ ID
    NO.: 26
    TNF-JK1 VH CDR- Residues 50-66 NINHDESEKYYVDSAKG
    H2 of SEQ ID
    NO.: 26
    TNF-JK1 VH CDR- Residues 99-115 IIRGRVGFDYYNYAMDV
    H3 of SEQ ID
    NO.: 26
    TNF-JK1 VL 27 DIRLTQSPSPLSASVGDRVTITC RASQSIG
    NYLN WYQHKPGKAPKLLIY AASSLQS GVPS
    RFSGTGSGTDFTLTISSLQPEDFATYYC QE
    SYSLI FAGGTKVEIK
    TNF-JK1 VL CDR- Residues 24-34 RASQSIGNYLN
    L1 of SEQ ID
    NO.: 27
    TNF-JK1 VL CDR- Residues 50-56 AASSLQS
    L2 of SEQ ID
    NO.: 27
    TNF-JK1 VL CDR- Residues 89-95 QESYSLI
    L3 of SEQ ID
    NO.: 27
    TNF-Y7C VH 28 EVQLVQSGAEVKKPGASVKVSCKTSGYTFS
    NYDIN WVRQPTGQGLEWMG WMDPNNGNTGY
    AQKFVG RVTMTRDTSKTTAYLELSGLKSED
    TAVYYCAR SSGSGGTWYKEYFQS WGQGTMV
    TVSS
    TNF-Y7C VH CDR- Residues 31-35 NYDIN
    H1 of SEQ ID
    NO.: 28
    TNF-Y7C VH CDR- Residues 50-66 WMDPNNGNTGYAQKFVG
    H2 of SEQ ID
    NO.: 28
    TNF-Y7C VH CDR- Residues 99-112 KSSGSGGTWYKEYFQS
    H3 of SEQ ID
    NO.: 28
    TNF-Y7C VL 29 DIVMTQSPLSLPVTPGEPASISC RSSQSLL
    HSNGYNYLD WYLQKPGQFPQLLIY LGSYRA
    S GVPDRFSGSGSGTDFTLKISRVEAEDVGV
    YYC MQRIEFPPGT FGQGTKLGIK
    TNF-Y7C VL CDR- Residues 24-39 RSSQSLLHSNGYNYLD
    L1 of SEQ ID
    NO.: 29
    TNF-Y7C VL CDR- Residues 55-61 LGSYRAS
    L2 of SEQ ID
    NO.: 29
    TNF-Y7C VL CDR- Residues 94-103 MQRIEFPPGT
    L3 of SEQ ID
    NO.: 29
    AE11-7 VH 30 EVQLVQSGAEVKKPGASVKVSCKTSGYSLT
    QYPIH WVRQAPGQRPEWMG WISPGNGNTKL
    SPKFQG RVTLSRDASAGTVFMDLSGLTSDD
    TAVYFCTS VDLGDH WGQGTLVTVSS
    AE11-7 VH CDR- Residues 31-35 QYPIH
    H1 of SEQ ID
    NO.: 30
    AE11-7 VH CDR- Residues 50-66 WISPGNGNTKLSPKFQG
    H2 of SEQ ID
    NO.: 30
    AE11-7 VH CDR- Residues 99-104 VDLGDH
    H3 of SEQ ID
    NO.: 30
    AE11-7 VL 31 DIVMTQSPEFQSVTPKEKVTITC RASQSIG
    SSLH WYQQKPDQSPKLLIN YASQSFS GVPS
    RFSGGGSGTDFTLTINSLEAEDAATYYC HQ
    SSNLPIT FGQGTRLEIK
    AE11-7 VL CDR- Residues 24-34 RASQSIGSSLH
    L1 of SEQ ID
    NO.: 31
    AE11-7 VL CDR- Residues 50-56 YASQSFS
    L2 of SEQ ID
    NO.: 31
    AE11-7 VL CDR- Residues 89-97 HQSSNLPIT
    L3 of SEQ ID
    NO.: 31
    AE11-13 VH 32 EVQLVESGGGLVQPGRSLRLSCAASGFTFD
    DYPMH WVRQAPGEGLEWVS GISSNSASIGY
    ADSVKG RFTISRDNAQNTLYLQMNSLGDED
    TAVYYCVS LTLG IGQGTLVTVSS
    AE11-13 VH CDR- Residues 31-35 DYPMH
    H1 of SEQ ID
    NO.: 32
    AE11-13 VH CDR- Residues 50-66 GISSNSASIGYADSVKG
    H2 of SEQ ID
    NO.: 32
    AE11-13 VH CDR- Residues 99-102 LTLG
    H3 of SEQ ID
    NO.: 32
    AE11-13 VL 33 DIRLTQSPSSLSASVGDRVTITC RASQSIG
    NYLH WYQQKPGKAPKLLIY AASSLQS GVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYC QQ
    SYSTLYS FGQGTKLEIK
    AE11-13 VL CDR- Residues 24-34 RASQSIGNYLH
    L1 of SEQ ID
    NO.: 33
    AE11-13 VL CDR- Residues 50-56 AASSLQS
    L2 of SEQ ID
    NO.: 33
    AE11-13 VL CDR- Residues 89-97 QQSYSTLYS
    L3 of SEQ ID
    NO.: 33
    • 1.2: Affinity Maturation of the Fully Human Anti-Human TNF Antibody AE11-5
  • The AE11-5 human antibody to human TNF was affinity matured by in vitro display technology. One light chain library was constructed to contain limited mutagenesis at the following residues: 28, 31, 32, 51, 55, 91, 92, 93, 95a and 96 (Kabat numbering). This library also contained framework germline back-mutations D1E, M4L, H11Q, R49K, H76N and Q103K as well as toggled residues at position 50(R/K) and 94(S/L) to allow for framework germlining during library selections. Two heavy chain libraries were made to contain limited mutagenesis in CDRH1 and CDRH2 at residues 30, 31, 33, 50, 52, and 55 to 58 (Kabat numbering) or in CDRH3 at residues 95 to 100b. The library containing CDRH1 and CDRH2 diversities also had framework germline back-mutations A18V and L64Q and toggled residue at 54(L/F) and 78(V/A). The CDRH3 library has an additional toggled residue at 100c(A/F).
  • All three libraries were selected separately for the ability to bind human or cynomolgus monkey TNF in the presence of decreasing concentrations of biotinylated human or cynomolgus monkey TNF antigens. All mutated CDR sequences recovered from library selections were recombined into additional libraries and the recombined libraries were subjected to more stringent selection conditions before individual antibodies are identified.
  • Table 11 provides a list of amino acid sequences of VH regions of affinity matured fully human TNF antibodies derived from AE11-5 Amino acid residues of individual CDRs of each VH sequence are indicated in bold.
  • TABLE 11
    List of amino acid sequences of affinity matured
    AE11-5 VH variants
    SEQ
    ID
    Clone NO: VH
    J685M2S2- 94 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    10VH ISWVRQAPGQGLEWMGGITPILGSANYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 95 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSYYS
    12VH ISWVRQAPGQGLEWMGGIMPILGTANYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 96 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    13VH ISWVRQAPGQGLEWMGGIIPILGSPIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 97 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    14VH ISWVRQAPGQGLEWMGGIIPILGSPIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 98 EVQLVQSGAEVKKPGSSVKVSCKASGGTFAWYS
    16VH ISWVRQAPGQGLEWMGGITPILGTANYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 99 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYA
    18VH ISWVRQAPGQGLEWMGGITPILGAATYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 100 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSIYA
    1VH ISWVRQAPGQGLEWMGGITPILGAAVYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 101 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    21VH ISWVRQAPGQGLEWMGGIMPILGTANYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 102 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    23VH ISWVRQAPGQGLEWMGGITPILGVAVYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 103 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    25VH ISWVRQAPGQGLEWMGGITPILGTANYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 104 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    27VH ISWVRQAPGQGLEWMGGITPILGSAHYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 105 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    28VH ISWVRQAPGQGLEWMGGITPILGSAIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 106 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    29VH ISWVRQAPGQGLEWMGGITPILGTAIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 107 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSIYT
    31VH ISWVRQAPGQGLEWMGGIIPILRNPIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 108 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSYYA
    32VH ISWVRQAPGQGLEWMGGIMPILGTPTYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 109 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSIYT
    35VH ISWVRQAPGQGLEWMGGIIPILGAPIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 110 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    37VH ISWVRQAPGQGLEWMGGITPILGSATYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 111 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYS
    38VH ISWVRQAPGQGLEWMGGIMPILGSASYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 112 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    43VH ISWVRQAPGQGLEWMGGIMPILGTASYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 113 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYS
    44VH ISWVRQAPGQGLEWMGGITPILGTANYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 114 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    45VH ISWVRQAPGQGLEWMGGIMPILGTATYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 115 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYT
    46VH ISWVRQAPGQGLEWMGGIMPILGSPHYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 116 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    47VH ISWVRQAPGQGLEWMGGITPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 117 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    48VH ISWVRQAPGQGLEWMGGIMPILGSATYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 118 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    4VH ISWVRQAPGQGLEWMGGIIPILGTPTYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 119 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    50VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J685M2S2- 120 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSLYT
    51VH ISWVRQAPGQGLEWMGGIMPILGAPRYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 121 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSYYA
    52VH ISWVRQAPGQGLEWMGGIMPILGSPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 122 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSIYA
    53VH ISWVRQAPGQGLEWMGGILPILGSPIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 123 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSIYA
    55VH ISWVRQAPGQGLEWMGGIIPILGSPIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 124 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    56VH ISWVRQAPGQGLEWMGGIVPILGAPLYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 125 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSIYA
    58VH ISWVRQAPGQGLEWMGGIMPILGAPIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 126 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSYYT
    5VH ISWVRQAPGQGLEWMGGIMPILGTPAYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 127 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYS
    61VH ISWVRQAPGQGLEWMGGITPILGAATYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 128 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    62VH ISWVRQAPGQGLEWMGGIIPILGTPTYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 129 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    63VH ISWVRQAPGQGLEWMGGIIPILGTPIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 130 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    64VH ISWVRQAPGQGLEWMGGITPILGIGNYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 131 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSIYA
    66VH ISWVRQAPGQGLEWMGGIVPILGAATYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 132 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    67VH ISWVRQAPGQGLEWMGGITPILGSSTYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 133 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    68VH ISWVRQAPGQGLEWMGGIMPILGTANYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 134 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    6VH ISWVRQAPGQGLEWMGGITPILGNSIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 135 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    70VH ISWVRQAPGQGLEWMGGITPILGSPIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 136 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    71VH ISWVRQAPGQGLEWMGGIMPILGTPTYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 137 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSYYA
    72VH ISWVRQAPGQGLEWMGGITPILGAANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 138 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    73VH ISWVRQAPGQGLEWMGGITPILGAAIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 139 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    75VH ISWVRQAPGQGLEWMGGITPILGTATYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 140 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYS
    76VH ISWVRQAPGQGLEWMGGITPILGSAHYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 141 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    77VH ISWVRQAPGQGLEWMGGITPILGNAIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 142 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    78VH ISWVRQAPGQGLEWMGGITPILRSAVYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 143 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYS
    7VH ISWVRQAPGQGLEWMGGIMPILGTANYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 144 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    80VH ISWVRQAPGQGLEWMGGITPILGTASYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 145 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    81VH ISWVRQAPGQGLEWMGGITPILGTAIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 146 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    82VH ISWVRQAPGQGLEWMGGITPILGSPAYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 147 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSRYA
    83VH ISWVRQAPGQGLEWMGGIIPILGPASYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 148 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    84VH ISWVRQAPGQGLEWMGGITPILDAAIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 149 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    86VH ISWVRQAPGQGLEWMGGIMPILGIPNYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 150 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSIYA
    87VH ISWVRQAPGQGLEWMGGITPILGSAIYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 151 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSYYA
    88VH ISWVRQAPGQGLEWMGGIMPILGTATYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 152 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    89VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYFDPKRADYWGQGTLVTVSS
    J685M2S2- 153 EVQLVQSGAEVKKPGSSVKVSCKASGGTFNWYT
    8VH ISWVRQAPGQGLEWMGGIMPILGTANYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 154 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    90VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYFDFTRADYWGQGTLVTVSS
    J685M2S2- 155 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    91VH ISWVRQAPGQGLEWMGGIIPILRFPTYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 156 EVQLVQSGAEVKKPGSSVKVSCKVSGGTFSWYS
    92VH ISWVRQAPGQGLEWMGGILPILDTANYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 157 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    93VH ISWVRQAPGQGLEWMGGIMPILGTAVYAQKFQG
    RVTITADESTSTAYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J685M2S2- 158 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSIYS
    94VH ISWVRQAPGQGLEWMGGILPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J688M2-11VH 159 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPTRADYWGQGTLVTVSS
    J688M2-13VH 160 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPSRADYWGQGTLVTVSS
    J688M2-14VH 161 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYFNPTRADYWGQGTLVTVSS
    J688M2-16VH 162 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPARFDYWGQGTLVTVSS
    J688M2-20VH 163 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYNPSRADYWGQGTLVTVSS
    J688M2-21VH 164 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPKRADYWGQGTLVTVSS
    J688M2-22VH 165 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPRRADYWGQGTLVTVSS
    J688M2-28VH 166 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    FYYDPTRADYWGQGTLVTVSS
    J688M2-29VH 167 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDFTRADYWGQGTLVTVSS
    J688M2-2VH 168 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYFDPKRADYWGQGTLVTVSS
    J688M2-37VH 169 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYFDPTRADYWGQGTLVTVSS
    J688M2-3VH 170 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPSRADYWGQGTLVTVSS
    J688M2-46VH 171 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARS
    LYYERTRADYWGQGTLVTVSS
    J688M2-48VH 172 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARW
    RFYIPIRFDYWGQGTLVTVSS
    J688M2-4VH 173 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDFTRADYWGQGTLVTVSS
    J688M2-50VH 174 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LFYDPSRADYWGQGTLVTVSS
    J688M2-52VH 175 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPVRADYWGQGTLVTVSS
    J688M2-56VH 176 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPIRADYWGQGTLVTVSS
    J688M2-57VH 177 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPKRADYWGQGTLVTVSS
    J688M2-58VH 178 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYNPIRFDYWGQGTLVTVSS
    J688M2-64VH 179 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYFDPARADYWGQGTLVTVSS
    J688M2-65VH 180 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VFFDPTRADYWGQGTLVTVSS
    J688M2-68VH 181 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VFYNPTRADYWGQGTLVTVSS
    J688M2-69VH 182 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYEGPSADYWGQGTLVTVSS
    J688M2-6VH 183 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYAPNRADYWGQGTLVTVSS
    J688M2-73VH 184 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LFYDPTRADYWGQGTLVTVSS
    J688M2-74VH 185 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYNPTRADYWGQGTLVTVSS
    J688M2-75VH 186 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPARADYWGQGTLVTVSS
    J688M2-7VH 187 EVQLVQSGAEVKKSGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPGRADYWGQGTLVTVSS
    J688M2-81VH 188 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYFDPSRADYWGQGTLVTVSS
    J688M2-82VH 189 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYFDPSRFDYWGQGTLVTVSS
    J688M2-83VH 190 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYFDFTRADYWGQGTLVTVSS
    J688M2-84VH 191 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPTRADYWGQGTLVTVSS
    J688M2-88VH 192 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYFDPSRADYWGQGTLVTVSS
    J688M2-89VH 193 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPSRFDYWGQGTLVTVSS
    J688M2-8VH 194 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    QYYDTSRADYWGQGTLVTVSS
    J688M2-90VH 195 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARS
    LYYDTTRFDYWGQGTLVTVSS
    J688M2-92VH 196 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VFYDPTRADYWGQGTLVTVSS
    J688M2-94VH 197 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPARADYWGQGTLVTVSS
    J688M2-95VH 198 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LFYDPRRADYWGQGTLVTVSS
    J688M2-96VH 199 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDTTRADYWGQGTLVTVSS
    J693FRM2S- 200 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    2L-32VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPARADYWGQGTLVTVSS
    J693FRM2S- 201 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    2L-40VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCSRG
    LYYDPTRADYWGQGTLVTVSS
    J693FRM2S- 202 EVQLVQSGAEVMKPGSSVKVSCKASGGTFSSYA
    2L-70VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCTRG
    LYYDPTRADYWGQGTLVTVSS
    J693FRM2-S 203 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYA
    2R-29VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J693FRM2S- 204 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    2R-46VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCTRG
    LYYDPTRADYWGQGTLVTVSS
    J693FRM2S- 205 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    2R-65VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCTRG
    IYYDPTRADYWGQGTLVTVSS
    J693M2S2L- 206 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    17VH ISWVRQAPGQGLEWMGGIIPILGTANYAQEFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2L- 207 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    32VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCVRG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2L- 208 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    67VH ISWVRQAPGQGLEWMGGIIPILGTASYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2L- 209 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    75VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCAKG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2L- 210 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    78VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCERG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2L- 211 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYA
    79VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2L- 212 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    94VH ISWVRQAPGQGLEWMGGIIPILGTANYAHKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2R- 213 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    22VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADCWGQGTLVTVSS
    J693M2S2R- 214 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    24VH ISWVQQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2R- 215 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    2VH ISWVRQAPGQGLEWMGGITPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2R- 216 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    31VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2R- 217 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    71VH TSWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2R- 218 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYA
    84VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFLG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J693M2S2R- 219 EVQLVQSGAEVKKPGSSVKVSCKASGGTSSSYA
    89VH ISWVRQAPGQGLEWMGGIIPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPTRADYWGQGTLVTVSS
    J703M1S3- 220 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    10VH ISWVRQAPGQGLEWMGGITPILGSATYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPKRADYWGQGTLVTVSS
    J703M1S3- 221 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    11VH ISWVRQAPGQGLEWMGGITPILGAASYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPTRADYWGQGTLVTVSS
    J703M1S3- 222 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    12VH ISWVRQAPGQGLEWMGGITPILGAASYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPARADYWGQGTLVTVSS
    J703M1S3- 223 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    13VH ISWVRQAPGQGLEWMGGITPILGAANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 224 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    14VH ISWVRQAPGQGLEWMGGIMPILGSPTYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPRRADYWGQGTLVTVSS
    J703M1S3- 225 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    16VH ISWVRQAPGQGLEWMGGITPILGSATYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPKRADYWGQGTLVTVSS
    J703M1S3- 226 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    17VH ISWVRQAPGQGLEWMGGIVPILGTPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 227 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    18VH ISWVRQAPGQGLEWMGGITPILGSANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPARADYWGQGTLVTVSS
    J703M1S3- 228 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    19VH ISWVRQAPGQGLEWMGGITPILGSPTYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 229 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    1VH ISWVRQAPGQGLEWMGGIMPILGTPVYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDFRRANYWGQGTLVTVSS
    J703M1S3- 230 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    20VH ISWVRQAPGQGLEWMGGITPILGAATYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPKRADYWGQGTLVTVSS
    J703M1S3- 231 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    21VH ISWVRQAPGQGLEWMGGITPILGDPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPKRADYWGQGTLVTVSS
    J703M1S3- 232 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    22VH ISWVRQAPGQGLEWMGGITPILGNPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDYKRADYWGQGTLVTVSS
    J703M1S3- 233 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    25VH ISWVRQAPGQGLEWMGGITPILGSANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LFYDFRRADYWGQGTLVTVSS
    J703M1S3- 234 EVQLVQSGAEVKKPGSSVKVSCKASGGTFAWYA
    28VH ISWVRQAPGQGLEWMGGITPILGNAIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 235 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    29VH ISWVRQAPGQGLEWMGGITPILGNPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 236 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    2VH TSWVRQAPGQGLEWMGGITPILGSPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDHRRADYWGQGTLVTVSS
    J703M1S3- 237 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    34VH ISWVRQAPGQGLEWMGGITPILGSPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDYKRADYWGQGTLVTVSS
    J703M1S3- 238 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    37VH ISWVRQAPGQGLEWMGGITPILGSAIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 239 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    38VH ISWVRQAPGQGLEWMGGITPILGTPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDFKRADYWGQGTLVTVSS
    J703M1S3- 240 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    3VH ISWVRQAPGQGLEWMGGIMPILGTPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPRRADYWGQGTLVTVSS
    J703M1S3- 241 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    41VH ISWVRQAPGQGLEWMGGITPILGSANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 242 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    42VH ISWVRQAPGQGLEWMGGITPILGAPVYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 243 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    45VH ISWVRQAPGQGLEWMGGITPILGSAIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 244 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    46VH ISWVRQAPGQGLEWMGGITPILGSPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPKRADYWGQGTLVTVSS
    J703M1S3- 245 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    47VH ISWVRQAPGQGLEWMGGIMPILGSANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPKRADYWGQGTLVTVSS
    J703M1S3- 246 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    4VH ISWVRQAPGQGLEWMGGITPILGNAIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPKRADYWGQGTLVTVSS
    J703M1S3- 247 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    50VH ISWVRQAPGQGLEWMGGITPILGAATYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 248 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    51VH ISWVRQAPGQGLEWMGGITPILGSPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDYRRADYWGQGTLVTVSS
    J703M1S3- 249 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    53VH ISWVRQAPGQGLEWMGGIMPILGIPTYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPARADYWGQGTLVTVSS
    J703M1S3- 250 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    54VH ISWVRQAPGQGLEWMGGITPILGSPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 251 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    57VH ISWVRQAPGQGLEWMGGITPILGSAVYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 252 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    5VH ISWVRQAPGQGLEWMGGITPILGSAIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDYKRADYWGQGTLVTVSS
    J703M1S3- 253 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    62VH ISWVRQAPGQGLEWMGGITPILGYPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 254 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    6VH ISWVRQAPGQGLEWMGGITPILGAATYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDFRRADYWGQGTLVTVSS
    J703M1S3- 255 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYP
    72VH ISWVRQAPGQGLEWMGGITPILGSAIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDFRRADYWGQGTLVTVSS
    J703M1S3- 256 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    78VH ISWVRQAPGQGLEWMGGITPILGTANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 257 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    79VH ISWVRQAPGQGLEWMGGITPILGSAVYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPKRADYWGQGTLVTVSS
    J703M1S3- 258 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    7VH ISWVRQAPGQGLEWMGGITPILGNPIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPARADYWGQGTLVTVSS
    J703M1S3- 259 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    81VH ISWVRQAPGQGLEWMGGIMPILGAPNYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDYTRADYWGQGTLVTVSS
    J703M1S3- 260 EVQLVQSGAEVKKPGSSVKVSCKASGGTFAWYA
    83VH ISWVRQAPGQGLEWMGGITPILGSPTYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 261 EVQLVQSGAEVKKPGSSVKVSCKASGGTFGWYA
    86VH TSWVRQAPGQGLEWMGGIIPILGTPNYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 262 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    87VH ISWVRQAPGQGLEWMGGIMPILGTPTYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDPKRADYWGQGTLVTVSS
    J703M1S3- 263 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYT
    88VH ISWVRQAPGQGLEWMGGIMPILGSPNYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    IYYDPKRADYWGQGTLVTVSS
    J703M1S3- 264 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    91VH ISWVRQAPGQGLEWMGGIMPILGSATYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYFDPKRADYWGQGTLVTVSS
    J703M1S3- 265 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYA
    93VH ISWVRQAPGQGLEWMGGITPILGAANYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    LYYDPKRADYWGQGTLVTVSS
    J703M1S3- 266 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSWYP
    9VH ISWVRQAPGQGLEWMGGITPILGAGIYAQKFQG
    RVTITADESTSTVYMELSSLRSEDTAVYYCARG
    VYYDFKRADYWGQGTLVTVSS
  • Table 12 provides a list of amino acid sequences of VL regions of affinity matured fully human TNF antibodies derived from AE11-5 Amino acid residues of individual CDRs of each VH sequence are indicated in bold.
  • TABLE 12
    List of amino acid sequences of affinity
    matured AE11-5 VL variants
    SEQ
    ID
    Clone NO: VL
    J685M2S2-17Vk 267 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPPPTF
    GQGTKVEIK
    J685M2S2-94Vk 268 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPPPTF
    GQWTKVEIK
    J688M2-37Vk 269 EIVLTQSPDFQSVTPKEKVTITCRARQSIGSSL
    HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTNFTLTINSLEAEDAATYYCHQSSSSPPPTF
    GQGTKVEIK
    J688M2-90Vk 270 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPPPTF
    GQGTKVEIK
    J693FRM2S2L- 271 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRSL
    26Vk HWYQQKPDQSPKLLIKHASQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNRSSPPSTF
    GQGTKVEIK
    J693FRM2S2L- 272 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRSL
    27Vk HWYQQKPDQSPKLLIKYASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPPVTF
    GQGTKVEIK
    J693FRM2S2L- 273 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    29Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRSNLPAPTF
    GQGTKVEIK
    J693FRM2S2L- 274 EIVLTQSPDFQSVTPKEKVTITCRASQIIGGSL
    39Vk HWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQPICSPPRTF
    GQGTKVEIK
    J693FRM2S2L- 275 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSNL
    3Vk HWYQQKPDQSPKLLIKYASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCSISPPATF
    GQGTKVEIK
    J693FRM2S2L- 276 EIVLTQSPDFQSVTPKEKVTITCRASQCIGTSL
    40Vk HWYQQKPDQSPKLLIKYDSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSSSPPPTF
    GQGTKVEIK
    J693FRM2S2L- 277 EIVLTQSPDFQSVTPKEKVTITCRASQNIGNSL
    42Vk HWYQQKPDQSPKLLIKYTSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQTSSLPLPTF
    GQGTKVEIK
    J693FRM2S2L- 278 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    43Vk HWYQQKPDQSPKLLIKYVSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQISDLPTSTF
    GQGTKVEIK
    J693FRM2S2L- 279 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSNL
    45Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGSSLPPPTF
    GQGTKVEIK
    J693FRM2S2L- 280 EIVLTQSPDFQSVTPKEKVTITCRASQCIGSSL
    46Vk HWYQQKPDQSPKLLIKHTSQSNSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSNSSPLSTF
    GQGTKVEIK
    J693FRM2S2L- 281 EIVLTQSPDFQSVTPKEKVTITCRASQNIGGSL
    47Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSSLPLPTF
    GQGTKVEIK
    J693FRM2S2L- 282 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    48Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSKSPPPTF
    GQGTKVEIK
    J693FRM2S2L- 283 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSCL
    52Vk HWYQQKPDQSPKLLIKYASQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRSSLPTPTF
    GQGTKVEIK
    J693FRM2S2L- 284 EIVLTQSPDFQSVTPKEKVTITCRASQSIGGRL
    53Vk HWYQQKPDQSPKLLIKYASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQASSSPSTTF
    GQGTKVEIK
    J693FRM2S2L- 285 EIVLTQSPDFQSVTPKEKVTITCRASQRIGPSL
    54Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSCLPSTTF
    GQGTKVEIK
    J693FRM2S2L- 286 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRSL
    58Vk HWYQQKPDQSPKLLIKYASQSRSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSGISPPTTF
    GQGTKVEIK
    J693FRM2S2L- 287 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTSL
    59Vk HWYQQKPDQSPKLLIKYVSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGMSSPAPTF
    GQGTKVEIK
    J693FRM2S2L- 288 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    5Vk HWYQQKPDQSPKLLIKYASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRRNSPPPTF
    GQGTKVEIK
    J693FRM2S2L- 289 EIVLTQSPDFQSVTPKEKVTITCRASQKIGSGL
    88Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNNSSPHKTF
    GQGTKVEIK
    J693FRM2S2L- 290 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSNL
    89Vk HWYQQKPDQSPKLLIKHSSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSSSPLPTF
    GQGTKVEIK
    J693FRM2S2L- 291 EIVLTQSPDFQSVTPKEKVTITCRASQNIGRSL
    8Vk HWYQQKPDQSPKLLIKYASQSSSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRSSPPPTF
    GQGTKVEIK
    J693FRM2S2L- 292 EIVLTQSPDFQSVTPKEKVTITCRASQCIGKSL
    90Vk HWYQQKPDQSPKLLIKHPSQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSIGLPPTTF
    GQGTKVEIK
    J693FRM2S2L- 293 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSSL
    91Vk HWYQQKPDQSPKLLIKHASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSISPPATF
    GQGTKVEIK
    J693FRM2S2L- 294 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSTL
    92Vk HWYQQKPDQSPKLLIKYESQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRCCSPTQTF
    GQGTKVEIK
    J693FRM2S2L- 295 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRKL
    94Vk HWYQQKPDQSPKLLIKYSSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSRSPPTTF
    GQGTKVEIK
    J693FRM2S2R- 296 EIVLTQSPDFQSVTPKEKVTITCRASQTIGTSL
    10Vk HWYQQKPDQSPKLLIKHASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPSPTF
    GQGTKVEIK
    J693FRM2S2R- 297 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    11Vk HWYQQKPDQSPKLLIKHVSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRGSSPPRTF
    GQGTKVEIK
    J693FRM2S2R- 298 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSTL
    12Vk HWYQQKPDQSPKLLIKHTSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRSSSPPPTF
    GQGTKVEIK
    J693FRM2S2R- 299 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSNL
    14Vk HWYQQKPDQSPKLLIKHGSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRHSSPRATF
    GQGTKVEIK
    J693FRM2S2R- 300 EIVLTQSPDFQSVTPKEKVTITCRASQKIGSNL
    15Vk HWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSSSPPATF
    GQGTKVEIK
    J693FRM2S2R- 301 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRSL
    16Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSRSPFRTF
    GQGTKVEIK
    J693FRM2S2R- 302 EIVLTQSPDFQSVTPKEKVTITCRASQCIGRRL
    34Vk HWYQQKPDQSPKLLIKHASQSRSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCTSSPPPTF
    GQGTKVEIK
    J693FRM2S2R- 303 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSNL
    36Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSLRLPPQTF
    GQGTKVEIK
    J693FRM2S2R- 304 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    39Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNRSLPRLTF
    GQGTKVEIK
    J693FRM2S2R- 305 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSCL
    3Vk HWYQQKPDQSPKLLIKYASQSISGVPSSSVASG
    SGTDFTLTINSLEAEDAATYYCHQRSSLPQPTF
    GQGTKVEIK
    J693FRM2S2R- 306 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRRL
    42Vk HWYQQKPDQSPKLLIKHPSQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSIDSPPPTF
    GQGTKVEIK
    J693FRM2S2R- 307 EIVLTQSPDFQSVTPKEKVTITCRASQTIGRSL
    45Vk HWYQQKPDQSPKLLIKYKSQSSSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRWGLPMPTF
    GQGTKVEIK
    J693FRM2S2R- 308 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSML
    48Vk HWYQQKPDQSPKLLIKHSSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQTNSLPPRTF
    GQGTKVEIK
    J693FRM2S2R- 309 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRSL
    50Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGSRSPLDTF
    GQGTKVEIK
    J693FRM2S2R- 310 EIVLTQSPDFQSVTPKEKVTITCRASQSIGCSL
    51Vk HWYQQKPDQSPKLLIKYASQSVSVVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSTLPPPTF
    GQGTKVEIK
    J693FRM2S2R- 311 EIVLTQSPDFQSVTPKEKVTITCRASQGIGTSL
    52Vk HWYQQKPDQSPKLLIKHDSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQTSSLPPPTF
    GQGTKVEIK
    J693FRM2S2R- 312 EIVLTQSPDFQSVTPKEKVTITCRASQIIGSSL
    56Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPLPTF
    GQGTKVEIK
    J693FRM2S2R- 313 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    58Vk HWYQQKPDQSPKLLIKYTSQSKSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGNRSPSTTF
    GQGTKVEIK
    J693FRM2S2R- 314 EIVLTQSPDFQSVTPKEKVTITCRASKRIGSSL
    59Vk HWYQQKPDQSPKLLIKHKSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRSASPPPTF
    GQGTKVEIK
    J693FRM2S2R- 315 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSSL
    5Vk HWYQQKPDQSPKLLIKHPSQSMSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSTSPPATF
    GQGTKVEIK
    J693FRM2S2R- 316 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    60Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRSSLPTPTF
    GQGTKVEIK
    J693FRM2S2R- 317 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSTL
    61Vk HWYQQKPDQSPKLLIKHASQSFSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSNCSPAHTF
    GQGTKVEIK
    J693FRM2S2R- 318 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSRL
    62Vk HWYQQKPDQSPKLLIKYVSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGSRLPPPTF
    GQGTKVEIK
    J693FRM2S2R- 319 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSTL
    63Vk HWYQQKPDQSPKLLIKHASQSNSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSCSPQATF
    GQGTKVEIK
    J693FRM2S2R- 320 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTSL
    64Vk HWYQQKPDQSPKLLIKYPSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSGRSPPHTF
    GQGTKVEIK
    J693FRM2S2R- 321 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    65Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSILPPPTF
    GQGTKVEIK
    J693FRM2S2R- 322 EIVLTQSPDFQSVTPKEKVTITCRASQCIGSYL
    92Vk HWYQQKPDQSPKLLIKHVSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPTLTF
    GQGTKVEIK
    J693FRM2S2R- 323 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    93Vk HWYQQKPDQSPKLLIKHASQSMSGVPSGFSGSG
    SGTDFTLTINSLEAEDAATYYCHQTNRSPPPTF
    GQGTKVEIK
    J693FRM2S2R- 324 EIVLTQSPDFQSVTPKEKVTITCRASQNIGTSL
    9Vk HWYQQKPDQSPKLLIKYVSQSISGVPSRFSGSG
    SGTDFTLNINSLEAEDAATYYCHQSSCLPRPTF
    GQGTKVEIK
    J693M2S2L- 325 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSPL
    10Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGSSSPPPTF
    GQGTKVEIK
    J693M2S2L- 326 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSTL
    11Vk HWYQQKPDQSPKLLIKHDSQSKSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSDSPAPTF
    GQGTKVEIK
    J693M2S2L- 327 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSCL
    12Vk HWYQQKPDQSPKLLIKHASQSNSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRISPLPTF
    GQGTKVEIK
    J693M2S2L- 328 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRRL
    13Vk HWYQQKPDQSPKLLIKHSSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCSSLPHPTF
    GQGTKVEIK
    J693M2S2L- 329 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSRL
    14Vk HWYQQKPDQSPKLLIKHASQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSCSSPLVTF
    GQGTKVEIK
    J693M2S2L- 330 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    16Vk HWYQQKPDQSPKLLIKHASQSSSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSGSSPQATF
    GQGTKVEIK
    J693M2S2L- 331 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    17Vk HWYQQKPDQSPKLLIKHASQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNRGSPPQTF
    GQGTKVEIK
    J693M2S2L- 332 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSIL
    18Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNTSLPPPTF
    GQGTKVEIK
    J693M2S2L- 333 EIVLTQSPDFQSVTPKEKVTITCRASQSIGNSL
    19Vk HWYQQKPDQSPKLLIKYPSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGSRLPVPTF
    GQGTKVEIK
    J693M2S2L-1Vk 334 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    HWYQQKPDQSPKLLIKHTSQSNSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPAPTF
    GQGTKVEIK
    J693M2S2L- 335 EIVLTQSPDFQSVTPKEKVTITCRASQNIGSSL
    20Vk HWYQQKPDQSPKLLIKHVSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSNSLPAPTF
    GQGTKVEIK
    J693M2S2L- 336 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    21Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSMSLPSATF
    GQGTKVEIK
    J693M2S2L- 337 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSSL
    22Vk HWYQQKPDQSPKLLIKHLSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQPCRLPPSTF
    GQGTKVEIK
    J693M2S2L- 338 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSLL
    23Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSCSSPRHTF
    GQGTKVEIK
    J693M2S2L- 339 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSSL
    24Vk HWYQQKPDQSPKLLIKHPSQSKSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSRSPAPTF
    GQGTKVEIK
    J693M2S2L- 340 EIVLTQSPDFQSVTPKEKVTITCRASQSIGGSL
    25Vk HWYQQKPDQSPKLLIKYSSQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSILPSLTF
    GQGTKVEIK
    J693M2S2L- 341 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    26Vk HWYQQKPDQSPKLLIKHPSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRNLPPRTF
    GQGTKVEIK
    J693M2S2L- 342 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSIL
    27Vk HWYQQKPDQSPKLLIKYGSQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNGSSPPRTF
    GQGTKVEIK
    J693M2S2L- 343 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    28Vk HWYQQKPDQSPKLLIKYFSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSCLPMQTF
    GQGTKVEIK
    J693M2S2L- 344 EIVLTQSPDFQSVTPKEKVTITCRASQNIGSSL
    29Vk HWYQQKPDQSPKLLIKYSSQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSISPPATF
    GQGTKVEIK
    J693M2S2L-2Vk 345 EIVLTQSPDFQSVTPKEKVTITCRASQCIGSSL
    HWYQQKPDQSPKLLIKHASQSNSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSTCLPPRTF
    GQGTKVEIK
    J693M2S2L- 346 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    30Vk HWYQQKPDQSPKLLIKYVSQSMSGVLSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQPSTSPRPTF
    GQGTKVEIK
    J693M2S2L- 347 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    31Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSSLPPSTF
    GQGTKVEIK
    J693M2S2L- 348 EIVLTQSPDFQSVTPKEKVTITCRASQSIGCSL
    32Vk HWYQQKPDQSPKLLIKYASQSNSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPSSTF
    GQGTKVEIK
    J693M2S2L- 349 EIVLTQSPDFQSVTPKEKVTITCRASQIIGTSL
    33Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSRSPPRTF
    GQGTKVEIK
    J693M2S2L- 350 EIVLTQSPDFQSVTPKEKVTITCRASQKIGTSL
    34Vk HWYQQKPDQSPKLLIKHESQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSGSPPPTF
    GQGTKVEIK
    J693M2S2L- 351 EIVLTQSPDFQSVTPKEKVTITCRASQTIGGSL
    35Vk HWYQQKPDQSPKLLIKHVSQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSISPPPTF
    GQGTKVEIK
    J693M2S2L- 352 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSDL
    36Vk HWYQQKPDQSPKLLIKHVSQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSCMSPSLTF
    GQGTKVEIK
    J693M2S2L- 353 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSNL
    37Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPNTTF
    GQGTKVEIK
    J693M2S2L- 354 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSIL
    38Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGRISPSSTF
    GQGTKVEIK
    J693M2S2L- 355 EIVLTQSPDFQSVTPKEKVTITCRASQSIGNRL
    39Vk HWYQQKPDQSPKLLIKHASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSGSLPTLTF
    GQGTKVEIK
    J693M2S2L-3Vk 356 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSSL
    HWYQQKPDQSPKLLIKHDSQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSSLPTHTF
    GQGTKVEIK
    J693M2S2L- 357 EIVLTQSPDFQSVTPKEKVTITCRASQTIGRSL
    40Vk HWYQQKPDQSPKLLIKHGSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRSSPPSTF
    GQGTKVEIK
    J693M2S2L- 358 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    41Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNCSSPPPTF
    GQGTKVEIK
    J693M2S2L- 359 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    44Vk HWYQQKPDQSPKLLIKYESQSDSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRRNSPPSTF
    GQGTKVEIK
    J693M2S2L- 360 EIVLTQSPDFQSVTPKEKVTITCRASQGIGSRL
    45Vk HWYQQKPDQSPKLLIKHGSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNRGLPAPTF
    GQGTKVEIK
    J693M2S2L- 361 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    46Vk HWYQQKPDQSPKLLIKYASQSSSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNHTSPPPTF
    GQGTKVEIK
    J693M2S2L- 362 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    47Vk HWYQQKPDQSPKLLIKHASQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSGRLPPPTF
    GQGTKVEIK
    J693M2S2L-4Vk 363 EIVLTQSPDFQSVTPKEKVTITCRASQYIGKRL
    HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSNISPPPTF
    GQGTKVEIK
    J693M2S2L- 364 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSSL
    51Vk HWYQQKPDQSPKLLIKHESQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPPPTF
    GQGTKVEIK
    J693M2S2L- 365 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSSL
    52Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRSSLPPSTF
    GQGTKVEIK
    J693M2S2L- 366 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRSL
    54Vk HWYQQKPDQSPKLLIKHPSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCSSSPAQTF
    GQGTKVEIK
    J693M2S2L- 367 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRSL
    55Vk HWYQQKPDQSPKLLIKHTSQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRSSLPLPTF
    GQGTKVEIK
    J693M2S2L- 368 EIVLTQSPDFQSVTPKEKVTITCRASQWIGSSL
    56Vk HWYQQKPDQSPKLLIKHTSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPPQTF
    GQGTKVEIK
    J693M2S2L- 369 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRSL
    58Vk HWYQQKPDQSPKLLIKYSSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGSSSPPPTF
    GQGTKVEIK
    J693M2S2L- 370 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    59Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSRLPPSTF
    GQGTKVEIK
    J693M2S2L-5Vk 371 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKYGSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNWSLPLPTF
    GQGTKVEIK
    J693M2S2L- 372 EIVLTQSPDFQSVTPKEKVTITCRASQRIGTSL
    62Vk HWYQQKPDQSPKLLIKYASQSKSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSCSPTPTF
    GQGTKVEIK
    J693M2S2L- 373 EIVLTQSPDFQSVTPKEKVTITCRASQSIGGSL
    64Vk HWYQQKPDQSPKLLIKYGSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRCVSPSPTF
    GQGTKVEIK
    J693M2S2L- 374 EIVLTQSPDFQSVTPKEKVTITCRASQSIGGTL
    65Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPARTF
    GQGTKVEIK
    J693M2S2L- 375 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    66Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCGSSPLHTF
    GQGTKVEIK
    J693M2S2L- 376 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTSL
    67Vk HWYQQKPDQSPKLLIKHPSQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSTSSPPPTF
    GQGTKVEIK
    J693M2S2L- 377 EIVLTQSPDFQSVTPKEKVTITCRASQNIGSSL
    68Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSGLPLPTF
    GQGTKVEIK
    J693M2S2L- 378 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRRL
    69Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGSSSPSPTF
    GQGTKVEIK
    J693M2S2L-6Vk 379 EIVLTQSPDFQSVTPKEKVTITCRASQRIGGNL
    HWYQQKPDQSPKLLIKHESQSNSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPSHTF
    GQGTKVEIK
    J693M2S2L- 380 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    70Vk HWYQQKPDQSPKLLIKYASQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCSSSPSHTF
    GQGTKVEIK
    J693M2S2L- 381 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    71Vk HWYQQKPDQSPKLLIKHASQSMSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRNSPPTTF
    GQGTKVEIK
    J693M2S2L- 382 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSRL
    72Vk HWYQQKPDQSPKLLIKHGSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSSSPPPTF
    GQGTKVEIK
    J693M2S2L- 383 EIVLTQSPDFQSVTPKEKVTITCRASQNIGSSL
    74Vk HWYQQKPDQSPKLLIKYASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSLLPAPTF
    GQGTKVEIK
    J693M2S2L- 384 EIVLTQSPDFQSVTPKEKVTITCRASQIIGTTL
    75Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSNLPPSTF
    GQGTKVEIK
    J693M2S2L- 385 EIVLTQSPDFQSVTPKEKVTITCRASQNIGGNL
    76Vk HWYQQKPDQSPKLLIKHASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSNLPPPTF
    GQGTKVEIK
    J693M2S2L- 386 EIVLTQSPDFQSVTPKEKVTITCRASQGIGGSL
    77Vk HWYQQKPDQSPKLLIKYASQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSACLPTRTF
    GQGTKVEIK
    J693M2S2L- 387 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTSL
    78Vk HWYQQKPDQSPKLLIKYASQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQIGSLPPPTF
    GQGTKVEIK
    J693M2S2R- 388 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRSL
    13Vk HWYQQKPDQSPKLLIKHASQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSRLPPPTF
    GQGTKVEIK
    J693M2S2R- 389 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    14Vk HWYQQKPDQSPKLLIKHNSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRSSSPPLTF
    GQGTKVEIK
    J693M2S2R- 390 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRNL
    15Vk HWYQQKPDQSPKLLIKHVSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRSRSPPSTF
    GQGTKVEIK
    J693M2S2R- 391 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    16Vk HWYQQKPDQSPKLLIKHASQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCSSLPAPTF
    GQGTKVEIK
    J693M2S2R- 392 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSSL
    17Vk HWYQQKPDQSPKLLIKHASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSRLPPQTF
    GQGTKVEIK
    J693M2S2R- 393 EIVLTQSPDFQSVTPKEKVTITCRASQCIGSRL
    18Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRGRLPPRTF
    GQGTKVEIK
    J693M2S2R- 394 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    19Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSTSLPRLTF
    GQGTKVEIK
    J693M2S2R- 395 EIVLTQSPDFQSVTPKEKVTITCRASQIIGSSL
    20Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRSSPQQTF
    GQGTKVEIK
    J693M2S2R- 396 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSTL
    21Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPPPTF
    GQGTKVEIK
    J693M2S2R- 397 EIVLTQSPDFQSVTPKEKVTITCRASQSIGNSL
    22Vk HWYQQKPDQSPKLLIKHGSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRRSSPRHTF
    GQGTKVEIK
    J693M2S2R- 398 EIVLTQSPDFQSVTPKEKVTITCRASQRIGRRL
    27Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSIGSPPLTF
    GQGTKVEIK
    J693M2S2R- 399 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRGL
    29Vk HWYQQKPDQSPKLLIKYGSQSMSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPPPTF
    GQGTKVEIK
    J693M2S2R-2Vk 400 EIVLTQSPDFQSVTPKEKVTITCRASQSIGCSL
    HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCTSLPLPTF
    GQGTKVEIK
    J693M2S2R- 401 EIVLTQSPDFQSVTPKEKVTITCRASQGIGSSL
    30Vk HWYQQKPDQSPKLLIKYVSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGSSLPTPTF
    GQGTKVEIK
    J693M2S2R- 402 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTSL
    31Vk HWYQQKPDQSPKLLIKHASQSSSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSRLPPLTF
    GQGTKVEIK
    J693M2S2R- 403 EIVLTQSPDFQSVTPKEKVTITCRASQVIGGVL
    32Vk HWYQQKPDQSPKLLIKYTSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPRPTF
    GQGTKVEIK
    J693M2S2R- 404 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    33Vk HWYQQKPDQSPKLLIKHSSQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRSNSPHRTF
    GQGTKVEIK
    J693M2S2R- 405 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRTL
    36Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCSISPQPTF
    GQGTKVEIK
    J693M2S2R- 406 EIVLTQSPDFQSVTPKEKVTITCRASQRIGNTL
    37Vk HWYQQKPDQSPKLLIKYPSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSGSSPPPTF
    GQGTKVEIK
    J693M2S2R- 407 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    39Vk HWYQQKPDQSPKLLIKYISQSMSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSCGLPPPTF
    GQGTKVEIK
    J693M2S2R-3Vk 408 EIVLTQSPDFQSVTPKEKVTITCRASQNIGTRL
    HWYQQKPDQSPKLLIKYGSQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRISPPPTF
    GQGTKVEIK
    J693M2S2R- 409 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSTL
    40Vk HWYQQKPDQSPKLLIKYVSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCSRLPPPTF
    GQGTKVEIK
    J693M2S2R- 410 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    44Vk HWYQQKPDQSPKLLIKYASQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSNLPSPTF
    GQGTKVEIK
    J693M2S2R- 411 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSNL
    45Vk HWYQQKPDQSPKLLIKHASQSMSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPRPTF
    GQGTKVEIK
    J693M2S2R- 412 EIVLTQSPDFQSVTPKEKVTITCRASQIIGSSL
    46Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSISSPSPTF
    GQGTKVEIK
    J693M2S2R- 413 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    47Vk HWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSNCLPPPTF
    GQGTKVEIK
    J693M2S2R- 414 EIVLTQSPDFQSVTPKEKVTITCRASQSIGKSL
    48Vk HWYQQKPDQSPKLLIKHESQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQANSLPPPTF
    GQGTKVEIK
    J693M2S2R-4Vk 415 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRRL
    HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCSSSPPSTF
    GQGTKVEIK
    J693M2S2R- 416 EIVLTQSPDFQSVTPKEKVTITCRASQIIGHSL
    52Vk HWYQQKPDQSPKLLIKHASQSILGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSIKSPPATF
    GQGTKVEIK
    J693M2S2R- 417 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTSL
    54Vk HWYQQKPDQSPKLLIKHTSQSKSGVPSRFSGSG
    SGTDFALTINSLEAEDAATYYCHQSSNSPRYTF
    GQGTKVEIK
    J693M2S2R- 418 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    55Vk HWYQQKPDQSPKLLIKHASQSHSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSGGSPPWTF
    GQGTKVEIK
    J693M2S2R- 419 EIVLTQSPDFQSVTPKEKVTITCRASQGIGRSL
    56Vk HWYQQKPDQSPKLLIKYASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSNRSPPPTF
    GQGTKVEIK
    J693M2S2R-5Vk 420 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTTL
    HWYQQKPDQSPKLLIKHVSQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPHPTF
    GQGTKVEIK
    J693M2S2R- 421 EIVLTQSPDFQSVTPKEKVTITCRASQIIGSSL
    60Vk HWYQQKPDQSPKLLIKYPSQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSWSSPLMTF
    GQGTKVEIK
    J693M2S2R- 422 EIVLTQSPDFQSVTPKEKVTITCRASQSIGNTL
    61Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPPPTF
    GQGTKVEIK
    J693M2S2R- 423 EIVLTQSPDFQSVTPKEKVTITCRASQRIGICL
    62Vk HWYQQKPDQSPKLLIKYASQSMSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGFSLPPATF
    GQGTKVEIK
    J693M2S2R- 424 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSCL
    63Vk HWYQQKPDQSPKLLIKYPSQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGSCSPTTTF
    GQGTKVEIK
    J693M2S2R- 425 EIVLTQSPDFQSVTPKEKVTITCRASQRIGNTL
    64Vk HWYQQKPDQSPKLLIKYPSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSGSSPPPTF
    GQGTKVEIK
    J693M2S2R- 426 EIVLTQSPDFQSVTPKEKVTITCRASQTIGTSL
    65Vk HWYQQKPDQSPKLLIKYASQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRCSLPPPTF
    GQGTKVEIK
    J693M2S2R- 427 EIVLTQSPDFQSVTPKEKVTITCRASQSIGGSL
    68Vk HWYQQKPDQSPKLLIKYASQSHSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCRISPRPTF
    GQGTKVEIK
    J693M2S2R- 428 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSSL
    69Vk HWYQQKPDQSPKLLIKHPSQSKSGVPSRFSGSG
    SGTDFTLSINSLEAEDAATYYCHQTSRSPLHTF
    GQGTKVEIK
    J693M2S2R-6Vk 429 EIVLTQSPDFQSVTPKEKVTITCRASQNIGKNL
    HWYQQKPDQSPKLLIKYPSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRSSPLSTF
    GQGTKVEIK
    J693M2S2R- 430 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    70Vk HWYQQKPDQSPKLLIKYMSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRVLPPPTF
    GQGTKVEIK
    J693M2S2R- 431 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    71Vk HWYQQKPDQSPKLLIKYGSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSISPRRTF
    GQGTKVEIK
    J693M2S2R- 432 EIVLTQSPDFQSVTPKEKVTITCRASQTIGRSL
    72Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRKSSPTPTF
    GQGTKVEIK
    J693M2S2R- 433 EIVLTQSPDFQSVTPKEKVTITCRASQRIGRQL
    75Vk HWYQQKPDQSPKLLIKHPSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPPQTF
    GQGTKVEIK
    J693M2S2R- 434 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    77Vk HWYQQKPDQSPKLLIKHTSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQICRSPSPTF
    GQGTKVEIK
    J693M2S2R- 435 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    78Vk HWYQQKPDQSPKLLIKYASQSSSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSGSPAPTF
    GQGTKVEIK
    J693M2S2R- 436 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    79Vk HWYQQKPDQSPKLLIKYSSQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQISSSPPPTF
    GQGTKVEIK
    J693M2S2R-7Vk 437 EIVLTQSPDFQSVTPKEKVTITCRASQTIGNSL
    HWYQQKPDQSPKLLIKHASQSNSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQTMTSPPPTF
    GQGTKVEIK
    J693M2S2R- 438 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    80Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRSSPSPTF
    GQGTKVEIK
    J693M2S2R- 439 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    81Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRRWSPPPTF
    GQGTKVEIK
    J693M2S2R- 440 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    82Vk HWYQQKPDQSPKLLIKYASQSNSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQISCLPLPTF
    GQGTKVEIK
    J693M2S2R- 441 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSSL
    83Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSISLPPPTF
    GQGTKVEIK
    J693M2S2R- 442 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRNL
    84Vk HWYQQKPDQSPKLLIKHTSQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQTSTLPPQTF
    GQGTKVEIK
    J693M2S2R- 443 EIVLTQSPDFQSVTPKEKVTITCRASQSIGRSL
    85Vk HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRNSPQPTF
    GQGTKVEIK
    J693M2S2R- 444 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTRL
    86Vk HWYQQKPDQSPKLLIKYVSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSHSPPPTF
    GQGTKVEIK
    J693M2S2R- 445 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSCL
    87Vk HWYQQKPDQSPKLLIKHRSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQWSSSPPPTF
    GQGTKVEIK
    J693M2S2R- 446 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSSL
    89Vk HWYQQKPDQSPKLLIKHPSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQTSGSPSHTF
    GQGTKVEIK
    J693M2S2R-8Vk 447 EIVLTQSPDFQSVTPKEKVTITCRASQGIGSSL
    HWYQQKPDQSPKLLIKYESQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPPPTF
    GQGTKVEIK
    J693M2S2R- 448 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    90Vk HWYQQKPDQSPKLLIKHDSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGSSSPPTTF
    GQGTKVEIK
    J693M2S2R- 449 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSNL
    91Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRISSPPSTF
    GQGTKVEIK
    J693M2S2R- 450 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSSL
    92Vk HWYQQKPDQSPKLLIKHASQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSCSSPPSTF
    GQGTKVEIK
    J693M2S2R- 451 EIVLTQSPDFQSVTPKEKVTITCRASQTIGSSL
    93Vk HWYQQKPDQSPKLLIKYVSQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQTISSPLPTF
    GQGTKVEIK
    J693M2S2R- 452 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    95Vk HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSCSPAHTF
    GQGTKVEIK
    J703M1S3-11Vk 453 EIVLTQSPDFQSVTPKEKVTITCRDSRCIGSNL
    HWYQQKPDQSPKLLIKHASQSSSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCSSSPPPTF
    GQGTKVEIK
    J703M1S3-13Vk 454 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSTL
    HWYQQKPDQSPKLLIKHASQSNSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPPPTF
    GQGTKVEIK
    J703M1S3-16Vk 455 EIVLTQSPDFQSVTPKEKVTITCRASQSIGDSL
    HWYQQKPDQSPKLLIKHASQSKSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGSTSPPRTF
    GQGTKVEIK
    J703M1S3-19Vk 456 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKHGSQSSSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSWSSPIPTF
    GQGTKVEIK
    J703M1S3-22Vk 457 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    HWYQQKPDQSPKLLIKYASQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSNLPSPTF
    GQGTKVEIK
    J703M1S3-26Vk 458 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    HWYQQKPDQSPKLLIKHASQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSGSSPPRTF
    GQGTKVEIK
    J703M1S3-29Vk 459 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRTSSPVRTF
    GQGTKVEIK
    J703M1S3-2Vk 460 EIVLTQSPDFQSVTPKEKVTITCRASQSIGNTL
    HWYQQKPDQSPKLLIKHVSQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQKVSSPSPTF
    GQGTKVEIK
    J703M1S3-30Vk 461 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSSL
    HWYQQKPDQSPKLLIKHASQSVSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSRSSPPPTF
    GQGTKVEIK
    J703M1S3-33Vk 462 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKHASQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPPSTF
    GQGTKVEIK
    J703M1S3-34Vk 463 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSSSPSTTF
    GQGTKVEIK
    J703M1S3-57Vk 464 EIVLTQSPDFQSVTPKEKVTITCRASQCIGSSL
    HWYQQKPDQSPKLLIKHESQSSSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRCTSPSPTF
    GQGTKVEIK
    J703M1S3-5Vk 465 EIVLTQSPDFQSVTPKEKVTITCRASQRIGSSL
    HWYQQKPDQSPKLLIKHPSQSDSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNCSLPLPTF
    GQGTKVEIK
    J703M1S3-62Vk 466 EIVLTQSPDFQSVTPKEKVTITCRASQCIGSSL
    HWYQQKPDQSPKLLIKHASQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQGISSPPQTF
    GQGTKVEIK
    J703M1S3-69Vk 467 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKHVSQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQRSSSPSPTF
    GQGTKVEIK
    J703M1S3-71Vk 468 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKHPSQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSIRLPPSTF
    GQGTKVEIK
    J703M1S3-78Vk 469 EIVLTQSPDFQSVTPKEKVTITCRANQSIGGSL
    HWYQQKPDQSPKLLIKHASQSKSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQDSRSPTRTF
    GQGTKVEIK
    J703M1S3-79Vk 470 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSGL
    HWYQQKPDQSPKLLIKHTSQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSLPHPTF
    GQGTKVEIK
    J703M1S3-7Vk 471 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKHASQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSSSPTPTF
    GQGTKVEIK
    J703M1S3-81Vk 472 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSRL
    HWYQQKPDQSPKLLIKYPSQSRSGVPSRFSGSG
    SGTDLTLTINSLEAEDAATYYCHQNGSLPPPTF
    GQGTKVEIK
    J703M1S3-82Vk 473 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSL
    HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSSSPPPTF
    GQGTKVEIK
    J703M1S3-86Vk 474 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSAL
    HWYQQKPDQSPKLLIKHASQSLSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQSSILPRPTF
    GQGTKVEIK
    J703M1S3-90Vk 475 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSNL
    HWYQQKPDQSPKLLIKHASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQTRTSPPLTF
    GQGTKVEIK
    J703M1S3-93Vk 476 EIVLTQSPDFQSVTPKEKVTITCRASQKIGSSL
    HWYQQKPDQSPKLLIKYGSQSTSGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQCISLPTPTF
    GQGTKVEIK
    J703M1S3-94Vk 477 EIVLTQSPDFQSVTPKEKVAITCRASQRIGSSL
    HWYQQKPDQSPKLLIKYASQSISGVPSRFSGSG
    SGTDFTLTINSLEAEDAATYYCHQNSSLPPPTF
    GQGTKVEIK
  • TABLE 13
    Amino acid residues observed in affinity matured AE11-5 antibodies
    AE11-5 Heavy chain variable region (SEQ ID NO: 1073)
    AE11-5VH 1234567890123456789012345678901234567890123456789012a345678901
    EVQLVQSGAEVKKPGSSAKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPILGTANYAQ
                     V           NW TTT              WT   FRSPI
                                 TY SV                M   TDAST
                                 GI P                 L   I NGS
                                 AN G                 V     P V
                                  F                   N     I H
                                  R                         V A
                                  L                         K R
                                                            F M
                                                              L
    234567890123456789012abc345678901234567890abc1234567890123
    KFLG RVTITADESTSTVYMELSSLRSEDTAVYYCAR GLYYDPTRADY WGQGTLVTVSS
      Q             A                   SVFFNTSWF
                                        WIVVEFASM
                                        TFP TRKP
                                        ARH IGRA
                                         Q  ADI
                                              Y
                                              V
                                              P
                                              N
                                              G
    AE11-5 Light chain variable region (SEQ ID NO: 1074)
    AE11-5VL 1234567890123456789012345678901234567890123456789012345678901
    DIVMTQSPDFHSVTPKEKVTITC RASQSIGSSLH WYQQKPDQSPKLLIR HASQSIS GVPSR
    E  L      Q                R  RR                KYV   L
                               T  TT                  P   V
                               N  GN                  T   T
                               I  NC                  G   S
                               C  KG                  S   M
                               G  CI                  E   N
                               K  HK                  D   K
                               Y  VM                      F
                               W  PL                      R
                                  LY
                                   P
                                   V
    2345678901234567890123456789012345a67890123456a
    FSGSGSGTDFTLTIHSLEAEDAATYYC HQSSSSPPPT FGQGTQVEIK
                  N              RRRL LS      K
                                 NGI  AR
                                 GIC  SL
                                 TCG  RT
                                 CNN  TA
                                 ITT  QQ
                                  MK  HH
                                       V
                                       M
  • TABLE 14
    Individual VH sequences from converted clones
    Protein Sequence
    region SEQ ID NO: 123456789012345678901234567890
    J703M1S3 478 EVQLVQSGAEVKKPGSSVKVSCKASGGTFS
    #2 WYATSWVRQAPGQGLEWMGGITPILGSPIY
    VH AQKFQGRVTITADESTSTVYMELSSLRSED
    TAVYYCARGVYYDHRRADYWGQGTLVTVSS
    J703M1S3 CDR-H1 Residues 31-35 WYATS
    #2 of SEQ ID
    VH NO.: 478
    J703M1S3 CDR-H2 Residues 50-66 GITPILGSPIYAQKFQG
    #2 of SEQ ID
    VH NO.: 478
    J703M1S3 CDR-H3 Residues 99-109 GVYYDHRRADY
    #2 of SEQ ID
    VH NO.: 478
    J703M1S3 479 EVQLVQSGAEVKKPGSSVKVSCKASGGTFS
    #13 WYAISWVRQAPGQGLEWMGGITPILGAANY
    VH AQKFQGRVTITADESTSTVYMELSSLRSED
    TAVYYCARGVYYDPKRADYWGQGTLVTVSS
    J703M1S3 CDR-H1 Residues 31-35 WYAIS
    #13 of SEQ ID
    NO.: 479
    J703M1S3 CDR-H2 Residues 50-66 GITPILGAANYAQKFQG
    #13 of SEQ ID
    VH NO.: 479
    J703M1S3 CDR-H3 Residues 99-109 GVYYDPKRADY
    #13 of SEQ ID
    VH NO.: 479
    J703M1S3 480 EVQLVQSGAEVKKPGSSVKVSCKASGGTFS
    #26 WYAISWVRQAPGQGLEWMGGITPILGTANY
    VH AQKFQGRVTITADESTSTVYMELSSLRSED
    TAVYYCARGVYYDPKRADYWGQGTLVTVSS
    J703M1S3 CDR-H1 Residues 31-35 WYAIS
    #26 of SEQ ID
    VH NO.: 480
    J703M1S3 CDR-H2 Residues 50-66 GITPILGTANYAQKFQG
    #26 of SEQ ID
    VH NO.: 480
    J703M1S3 CDR-H3 Residues 99-109 GVYYDPKRADY
    #26 of SEQ ID
    VH NO.: 480
    J703M1S3 481 EVQLVQSGAEVKKPGSSVKVSCKASGGTFS
    #30 WYAISWVRQAPGQGLEWMGGITPILGSPIY
    VH AQKFQGRVTITADESTSTVYMELSSLRSED
    TAVYYCARGVYYDPKRADYWGQGTLVTVSS
    J703M1S3 CDR-H1 Residues 31-35 WYAIS
    #30 of SEQ ID
    VH NO.: 481
    J703M1S3 CDR-H2 Residues 50-66 GITPILGSPIYAQKFQG
    #30 of SEQ ID
    VH NO.: 481
    J703M1S3 CDR-H3 Residues 99-109 GVYYDPKRADY
    #30 of SEQ ID
    VH NO.: 481
    J703M1S3 482 EVQLVQSGAEVKKPGSSVKVSCKASGGTFS
    #33 WYPISWVRQAPGQGLEWMGGITPILGAGIY
    VH AQKFQGRVTITADESTSTVYMELSSLRSED
    TAVYYCARGVYYDFKRADYWGQGTLVTVSS
    J703M1S3 CDR-H1 Residues 31-35 WYPIS
    #33 of SEQ ID
    VH NO.: 482
    J703M1S3 CDR-H2 Residues 50-66 GITPILGAGIYAQKFQG
    #33 of SEQ ID
    VH NO.: 482
    J703M1S3 CDR-H3 Residues 99-109 GVYYDFKRADY
    #33 of SEQ ID
    VH NO.: 482
    J703M1S3 483 EVQLVQSGAEVKKPGSSVKVSCKASGGTFS
    #35 WYAISWVRQAPGQGLEWMGGITPILGSATY
    VH AQKFQGRVTITADESTSTVYMELSSLRSED
    TAVYYCARGIYYDPKRADYWGQGTLVTVSS
    J703M1S3 CDR-H1 Residues 31-35 WYAIS
    #35 of SEQ ID
    VH NO.: 483
    J703M1S3 CDR-H2 Residues 50-66 GITPILGSATYAQKFQG
    #35 of SEQ ID
    VH NO.: 483
    J703M1S3 CDR-H3 Residues 99-109 GIYYDPKRADY
    #35 of SEQ ID
    VH NO.: 483
    J703M1S3 484 EVQLVQSGAEVKKPGSSVKVSCKASGGTFS
    #38 WYAISWVRQAPGQGLEWMGGITPILGTPIY
    VH AQKFQGRVTITADESTSTVYMELSSLRSED
    TAVYYCARGVYYDFKRADYWGQGTLVTVSS
    J703M1S3 CDR-H1 Residues 31-35 WYAIS
    #38 of SEQ ID
    VH NO.: 484
    J703M1S3 CDR-H2 Residues 50-66 GITPILGTPIYAQKFQG
    #38 of SEQ ID
    VH NO.: 484
    J703M1S3 CDR-H3 Residues 99-109 GVYYDFKRADY
    #38 of SEQ ID
    VH NO.: 484
    J703M1S3 485 EVQLVQSGAEVKKPGSSVKVSCKASGGTFS
    #69 WYAISWVRQAPGQGLEWMGGITPILGSPIY
    VH AQKFQGRVTITADESTSTVYMELSSLRSED
    TAVYYCARGIYYDPKRADYWGQGTLVTVSS
    J703M1S3 CDR-H1 Residues 31-35 WYAIS
    #69 of SEQ ID
    VH NO.: 485
    J703M1S3 CDR-H2 Residues 50-66 GITPILGSPIYAQKFQG
    #69 of SEQ ID
    VH NO.: 485
    J703M1S3 CDR-H3 Residues 99-109 GIYYDPKRADY
    #69 of SEQ ID
    VH NO.: 485
    J703M1S3 486 EVQLVQSGAEVKKPGSSVKVSCKASGGTFS
    #90 WYAISWVRQAPGQGLEWMGGITPILGSPIY
    VH AQKFQGRVTITADESTSTVYMELSSLRSED
    TAVYYCARGVYYDYKRADYWGQGTLVTVSS
    J703M1S3 CDR-H1 Residues 31-35 WYAIS
    #90 of SEQ ID
    VH NO.: 486
    J703M1S3 CDR-H2 Residues 50-66 GITPILGSPIYAQKFQG
    #90 of SEQ ID
    VH NO.: 486
    J703M1S3 CDR-H3 Residues 99-109 GVYYDYKRADY
    #90 of SEQ ID
    VH NO.: 486
  • TABLE 15
    Individual clones VL sequences
    Protein Sequence
    region 123456789012345678901234567890
    J703M1S3 487 EIVLTQSPDFQSVTPKEKVTITCRASQSIG
    #2 NTLHWYQQKPDQSPKLLIKHVSQSVSGVPS
    VL RFSGSGSGTDFTLTINSLEAEDAATYYCHQ
    KVSSPSPTFGQGTKVEIK
    J703M1S3 CDR-L1 Residues 24-34 RASQSIGNTLH
    #2 of SEQ ID
    VL NO.: 487
    J703M1S3 CDR-L2 Residues 50-56 HVSQSVS
    #2 of SEQ ID
    VL NO.: 487
    J703M1S3 CDR-L3 Residues 89-98 HQKVSSPSPT
    #2 of SEQ
    VL ID NO.: 487
    J703M1S3 488 EIVLTQSPDFQSVTPKEKVTITCRASQSIG
    #13 STLHWYQQKPDQSPKLLIKHASQSNSGVPS
    VL RFSGSGSGTDFTLTINSLEAEDAATYYCHQ
    SSSLPPPTFGQGTKVEI
    J703M1S3 CDR-L1 Residues 24-34 RASQSIGSTLH
    #13 of SEQ ID
    VL NO.: 488
    J703M1S3 CDR-L2 Residues 50-56 HASQSNS
    #13 of SEQ ID
    VL NO.: 488
    J703M1S3 CDR-L3 Residues 89-98 HQSSSLPPPT
    #13 of SEQ
    VL ID NO.: 488
    J703M1S3 489 EIVLTQSPDFQSVTPKEKVTITCRASQSIG
    #26 SRLHWYQQKPDQSPKLLIKHASQSTSGVPS
    VL RFSGSGSGTDFTLTINSLEAEDAATYYCHQ
    SGSSPPRTFGQGTKVEIK
    J703M1S3 CDR-L1 Residues 24-34 RASQSIGSRLH
    #26 of SEQ ID
    VL NO.: 489
    J703M1S3 CDR-L2 Residues 50-56 HASQSTS
    #26 of SEQ ID
    VL NO.: 489
    J703M1S3 CDR-L3 Residues 89-98 HQSGSSPPRT
    #26 of SEQ
    VL ID NO.: 489
    J703M1S3 490 EIVLTQSPDFQSVTPKEKVTITCRASQRIG
    #30 SSLHWYQQKPDQSPKLLIKHASQSVSGVPS
    VL RFSGSGSGTDFTLTINSLEAEDAATYYCHQ
    SRSSPPPTFGQGTKVEIK
    J703M1S3 CDR-L1 Residues 24-34 RASQRIGSSLH
    #30 of SEQ ID
    VL NO.: 490
    J703M1S3 CDR-L2 Residues 50-56 HASQSVS
    #30 of SEQ ID
    VL NO.: 490
    J703M1S3 CDR-L3 Residues 89-98 HQSRSSPPPT
    #30 of SEQ
    VL ID NO.: 490
    J703M1S3 491 EIVLTQSPDFQSVTPKEKVTITCRASQSIG
    #33 SSLHWYQQKPDQSPKLLIKHASQSTSGVPS
    VL RFSGSGSGTDFTLTINSLEAEDAATYYCHQ
    SSSSPPSTFGQGTKVEIK
    J703M1S3 CDR-L1 Residues 24-34 RASQSIGSSLH
    #33 of SEQ ID
    VL NO.: 491
    J703M1S3 CDR-L2 Residues 50-56 HASQSTS
    #33 of SEQ ID
    VL NO.: 491
    J703M1S3 CDR-L3 Residues 89-98 HQSSSSPPST
    #33 of SEQ
    VL ID NO.: 491
    J703M1S3 492 EIVLTQSPDFQSVTPKEKVTITCRASQTIG
    #35 SSLHWYQQKPDQSPKLLIKHASQSISGVPS
    VL RFSGSGSGTDFTLTINSLEAEDAATYYCHQ
    TSSLPTPTFGQGTKVEIK
    J703M1S3 CDR-L1 Residues 24-34 RASQTIGSSLH
    #35 of SEQ ID
    VL NO.: 492
    J703M1S3 CDR-L2 Residues 50-56 HASQSIS
    #35 of SEQ ID
    VL NO.: 492
    J703M1S3 CDR-L3 Residues 89-98 HQTSSLPTPT
    #35 of SEQ
    VL ID NO.: 492
    J703M1S3 493 EIVLTQSPDFQSVTPKEKVTITCRASQTIG
    #38 SSLHWYQQKPDQSPKLLIKHASQSISGVPS
    VL RFSGSGSGTDFTLTINSLEAEDAATYYCHQ
    SSSSPPPTFGQGTKVEIK
    J703M1S3 CDR-L1 Residues 24-34 RASQTIGSSLH
    #38 of SEQ ID
    VL NO.: 493
    J703M1S3 CDR-L2 Residues 50-56 HASQSIS
    #38 of SEQ ID
    VL NO.: 493
    J703M1S3 CDR-L3 Residues 89-98 HQSSSSPPPT
    #38 of SEQ
    VL ID NO.: 493
    J703M1S3 494 EIVLTQSPDFQSVTPKEKVTITCRASQSIG
    #69 SSLHWYQQKPDQSPKLLIKHVSQSLSGVPS
    VL RFSGSGSGTDFTLTINSLEAEDAATYYCHQ
    RSSSPSPTFGQGTKVEIK
    J703M1S3 CDR-L1 Residues 24-34 RASQSIGSSLH
    #69 of SEQ ID
    VL NO.: 494
    J703M1S3 CDR-L2 Residues 50-56 HVSQSLS
    #69 of SEQ ID
    VL NO.: 494
    J703M1S3 CDR-L3 Residues 89-98 HQRSSSPSPT
    #69 of SEQ
    VL ID NO.: 494
    J703M1S3 495 EIVLTQSPDFQSVTPKEKVTITCRASQSIG
    #90 SNLHWYQQKPDQSPKLLIKHASQSISGVPS
    VL RFSGSGSGTDFTLTINSLEAEDAATYYCHQ
    TRTSPPLTFGQGTKVEIK
    J703M1S3 CDR-L1 Residues 24-34 RASQSIGSNLH
    #90 of SEQ ID
    VL NO.: 495
    J703M1S3 CDR-L2 Residues 50-56 HASQSIS
    #90 of SEQ ID
    VL NO.: 495
    J703M1S3 CDR-L3 Residues 89-98 HQTRTSPPLT
    #90 of SEQ
    VL ID NO.: 495
  • TABLE 16
    AE11-5 affinity matured scFv clones converted to full length IgG
    Full length
    ScFv IgG (protein)
    clone name HC plasmid LC plasmid name
    J703M1S3#2 pJP368; pHybE-hCg1,z,non- pJP369; pHybE-hCk V3- AE11-5 AM1
    a,mut(234,235)-J703M1S3#2 J703M1S31#2
    J703M1S3#13 pJP370; pHybE-hCg1,z,non- pJP371; pHybE-hCk V3- AE11-5 AM2
    a,mut(234,235)-J703M1S3#13 J703M1S3#13
    J703M1S3#26 pJP372; pHybE-hCg1,z,non- pJP373; pHybE-hCk V3- AE11-5 AM3
    a,mut(234,235)-J703M1S3#26 J703M1S3#26
    J703M1S3#30 pJP374; pHybE-hCg1,z,non- pJP375; pHybE-hCk V3- AE11-5 AM4
    a,mut(234,235)-J703M1S3#30 J703M1S3#30
    J703M1S3#33 pJP376; pHybE-hCg1,z,non- pJP377; pHybE-hCk V3- AE11-5 AM5
    a,mut(234,235)-J703M1S3#33 J703M1S3#33
    J703M1S3#35 pJP378; pHybE-hCg1,z,non- pJP379; pHybE-hCk V3- AE11-5 AM6
    a,mut(234,235)-J703M1S3#35 J703M1S3#35
    J703M1S3#38 pJP382; pHybE-hCg1,z,non- pJP383; pHybE-hCk V3- AE11-5 AM8
    a,mut(234,235)-J703M1S3#38 J703M1S3#38
    J703M1S3#69 pJP384; pHybE-hCg1,z,non- pJP385; pHybE-hCk V3- AE11-5 AM9
    a,mut(234,235)-J703M1S3#69 J703M1S3#69
    J703M1S3#90 pJP386; pHybE-hCg1,z,non- pJP387; pHybE-hCk V3- AE11-5 AM10
    a,mut(234,235)-J703M1S3#90 J703M1S3#90
    • 1.3 TNF Enzyme-Linked Immunosorbent Assay Protocol (ELISA) and Assay Result
  • The following protocol is used to characterize the binding of TNF antibodies to biotinylated human or cyno TNF by enzyme-linked immunosorbent assay (ELISA). An ELISA plate was coated with 50 μl per well of goat anti human IgG-Fc at 2 μg/ml, overnight at 4° C. The plate was washed 3 times with PBS/Tween. 50 μl Mab diluted to 1 μg/ml in PBS/0.1% BSA was added to appropriate wells and incubated for 1 hour at room temperature (RT). The plate was washed 3 times with PBS/Tween. 50 μl of serial diluted biotin-human TNF was added to appropriate wells and incubated for 1 hour at RT. The plate was washed 3 times with PBS/Tween. 50 μl of streptavidin-HRP diluted 1:10,000 in PBS/0.1% BSA was added to appropriate wells and incubated for 1 hour at RT. The plate was washed 3 times with PBS/Tween. 50 μl of TMB was added to appropriate wells and the reaction was allowed to proceed for 1 minute. The reaction was stopped with 50 μl/well 2N H2SO4 and the absorbance read at 450 nm. Results are shown in Table 17.
  • TABLE 17
    EC50 in hTNF EC50 in cynoTNF
    IgG Name ELISA (nM) ELISA (nM)
    AE11-5-AM1 1.06 2.14
    AE11-5-AM2 522.5 >845
    AE11-5-AM3 1.57 1.55
    AE11-5-AM4 18.32 750.3
    AE11-5-AM5 17.7 2.2
    AE11-5-AM6 1.37 >720
    AE11-5-AM7 10.32 1.26
    AE11-5-AM8 250.2 58.58
    AE11-5-AM9 16.72 5.29
    AE11-5-AM10 0.98 0.28
    • 1.4 TNF Neutralization Potency of TNF Antibodies by L929 Bioassay
  • Human TNF was prepared at Abbott Bioresearch Center (Worcester, Mass., US) and received from the Biologics Pharmacy. Mouse TNF was prepared at Abbott Bioresearch Center and received from the Biologics Pharmacy. Rat TNF was prepared at Abbott Bioresearch Center and received from the Biologics Pharmacy. Rabbit TNF was purchased from R&D Systems. Rhesus/Macaque TNF (rhTNF) was purchased from R&D Systems. Actinomycin was purchased from Sigma Aldrich and resuspended at a stock concentration of 10 mg/mL in DMSO.
  • Assay Media: 10% FBS (Hyclone#SH30070.03), Gibco reagents: RPMI 1640 (#21870), 2 mM L-glutamine (#25030), 50 units/mL penicillin/50 μg/mL streptomycin (#15140), 0.1 mM MEM non-essential amino acids (#11140) and 5.5×10−5 M 2-mercaptoethanol (#21985-023).
  • L929 cells were grown to a semi-confluent density and harvested using 0.05% tryspin (Gibco#25300). The cells were washed with PBS, counted, and resuspended at 1E6 cells/mL in assay media containing 4 μg/mL actinomycin D. The cells were seeded in a 96-well plate (Costar#3599) at a volume of 50 μL and 5E4 cells/well. Wells received 50 μL of assay media, bringing the volume to 100 μL.
  • A test sample was prepared as follows. The test and control IgG proteins were diluted to a 4× concentration in assay media and serial 1:3 dilutions were performed. TNF species were diluted to the following concentrations in assay media: 400 pg/mL huTNF, 200 pg/mL muTNF, 600 pg/mL ratTNF, and 100 pg/mL rabTNF. Antibody sample (200 μL) was added to the TNF (200 μL) in a 1:2 dilution scheme and allowed to incubate for 0.5 hour at room temperature.
  • To measure huTNF neutralization potency in this assay, the antibody/TNF solution was added to the plated cells at 100 μL for a final concentration at 375 nM-0.019 nM. The final concentration of TNF was as follows: 100 pg/mL huTNF, 50 pg/mL muTNF, 150 pg/mL ratTNF, and 25 pg/mL rabTNF. The plates were incubated for 20 hours at 37° C., 5% CO2. To quantitate viability, 100 μL was removed from the wells and 10 μL of WST-1 reagent (Roche cat#11644807001) was added. Plates were incubated under assay conditions for 3.5 hours, centrifuged at 500×g, and 75 μL of supernatant transferred to an ELISA plate (Costar cat#3369). The plates were read at OD 420-600 nm on a Spectromax 190 ELISA plate reader. The neutralization potency of selected TNF/IL-17 DVD-Ig binding proteins is shown in Table 18.
  • TABLE 18
    hu TNF neutralization rhesus TNF neutralization IC50
    IgG Name IC50 (nM) (nM)
    AE11-5 AM1 0.439 0.251
    AE11-5 AM2 1.241 0.756
    AE11-5 AM3 0.291 0.165
    AE11-5 AM4 0.259 0.109
    AE11-5 AM5 0.968 0.613
    AE11-5 AM6 2.029 0.652
    AE11-5 AM7 0.049 0.104
    AE11-5 AM8 1.356 3.040
    AE11-5 AM9 0.391 0.123
    AE11-5 AM10 0.678 0.140
    • Example 2 Affinity Maturation of a Humanized Anti-Human TNF Antibody hMAK-195
  • The mouse anti-human TNF antibody MAK-195 was humanized and affinity-matured to generate a panel of humanized MAK195 variants that have cross-reactivity to cyno-TNF and improved affinity and binding kinetics against both human and cyno TNF.
  • To improve the affinity of hMAK195 to TNF, hypermutated CDR residues were identified from other human antibody sequences in the IgBLAST database that also shared high identity to germlines VH3-53 and IGKV1-39. The corresponding hMAK195 CDR residues were then subjected to limited mutagenesis by PCR with primers having low degeneracy at these positions to create three antibody libraries in the scFv format. The first library contained mutations at residues 31, 32, 33, 35, 50, 52, 53, 54, 56 and 58 in the VH CDR1 and 2 (Kabat numbering); the second library at residues 95 to 100, 100a, 101, and 102 in VH CDR3; and the third library at residues 28, 30, 31, 32, 50, 53, 92, 93, 94, and 95 in the three VL CDRs. To further increase the identity of hMAK195 to the human germline framework sequences, a binary degeneracy at VH positions 60 (D/A), 61 (S/D), 62 (T/S), 63 (L/V), and 65 (S/G) were introduced into the first library. Also, a binary degeneracy at VL positions 24 (K/R), 33 (V/L), 54 (R/L), 55 (H/Q), 56 (T/S), 91 (H/S) and 96 (F/Y) were introduced into the third library.
  • These hMAK195 variants were selected against a low concentration of biotinylated TNF for improved on-rate, off-rate, or both were carried out and antibody protein sequences of affinity-modulated hMAK195 were recovered for converting back to IgG for further characterization. All three libraries were selected separately for the ability to bind human or cynomolgus monkey TNF in the presence of decreasing concentrations of biotinylated human or cynomolgus monkey TNF antigens. All mutated CDR sequences recovered from library selections were recombined into additional libraries and the recombined libraries were subjected to more stringent selection conditions before individual antibodies are identified.
  • Table 19 provides a list of amino acid sequences of VH and VL of the humanized MAK-195 which were subjected to the affinity maturation selection protocol Amino acid residues of individual CDRs of each VH and VL sequence are indicated in bold.
  • TABLE 19
    List of amino acid sequences of affinity
    matured hMAK195 VH variants
    SEQ ID
    Clone NO: VH
    rHC1_B8 496 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSIIRGDGSTDYASTLKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC1_H12 497 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSIIRGDGSTDYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_E1 498 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYGVNWVRQAPGK
    GLEWVSIIWGDGATDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_A2 499 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMISSDGFTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC1_H6 500 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIAADGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    H1 + H2_D7 501 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRADGSTDYASSLKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_D9 502 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRDDGSTDYADTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_A10 503 EVQLVESGGGLVQPGGSLRLSCAASGETFSHIGVSWVRQAPGK
    GLEWVSMISYAGSTDYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARLLHKGPIDYWGQGTLVTVSS
    H1 + H2_A5 504 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFGVNWVRQAPGK
    GLEWVSMIWSDGSTDYADTVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_F8 505 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSIIRADGSTDYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_D1 506 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVQWVRQAPGK
    GLEWVSMIRGDGSTDYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARPSHHGLIDNWGQGTLVTVSS
    rHC2_C2 507 EVQLVESGGGLVQPGGSLRLSCAASGFTFSELGVNWVRQAPGK
    GLEWVSYISDVGSTYYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARDWHHGRFDYWGQGTLVTVSS
    rHC1_G4 508 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSLIRADGSTDYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_F3 509 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRADGFTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARDWQHGPSVYWGQGTLVTVSS
    rHC1_B4 510 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSIIRADGVTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_G3 511 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVHWVRQAPGK
    GLEWVSMIGADGYTDYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_D7 512 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMISADGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_D5 513 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRSDGFTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    TEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_E4 514 EVQLVESGGGLVQPGGSLRLSCAASGFTFSEYGVNWVRQAPGK
    GLEWVSIIWHDGSTAYADTVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_E10 515 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSLIRGDGSTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_B6 516 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGVSWVRQAPGK
    GLEWVSMIWGDGSTDYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_B7 517 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRDDGSTYYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLGYWGQGTLVTVSS
    H1 + H2_G8 518 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFGVNWVRQAPGK
    GLEWVSMIWAGGSTAYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_G5 519 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSLIGADGSTDYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQYGPLAYWGQGTLVTVSS
    H1 + H2_F1 520 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIEGDGGTHYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC19 521 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWADGSTHYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPAAYWGQGTLVTVSS
    H1 + H2_A10 522 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAXGK
    GLEWVSMISADGTTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_B9 523 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSIIRGDGTTDYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLGYWGQGTLVTVSS
    H1 + H2_F7 524 EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYGVGWVRQAPGK
    GLEWVSMIWGAGSTNYADTVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_B1 525 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGVNWVRQAPGK
    GLEWVSMIWADGTTDYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_H9 526 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSVIGGDGYTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    H1 + H2_A12 527 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGX
    GLEWVSMISSDGYTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC2_G8 528 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMIWSDGSTHYADTVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC2_B4 529 EVQLVESGGGLVQPGGSLRLSCAASGFTFSQLGVTWVRQAPGK
    GLEWVSTISDAGSTYYASSVKGRFTIIRINSKNTLYLQMNSLR
    AEDTAVYYCARDWHHGRFAYWGQGTLVTVSS
    H1 + H2_G5 530 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSIIRGDGSTYYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_C6 531 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVQWVRQAPGK
    GLEWVSMIRDDGSTSYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_F5 532 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSIIRGDGSTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    H1 + H2_B4 533 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYGVNWVRQAPGK
    GLEWVSMISGDGSTDYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_F6 534 EVQLVESGGGLVQPGGSLRLSCAASGFTFSHFGVTWVRQAPGK
    GLEWVSNIWASGSTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_B6 535 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRADGSTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVGYWGQGTLVTVSS
    H1 + H2_A3 536 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGVNWVRQAPGK
    GLEWVSVIWGDGSTAYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_D10 537 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSIIRGDGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    rHC18 538 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWSDGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-18 539 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWADGSTHYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    rHC2_E6 540 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSLIRGDGSTDYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_D4 541 EVQLVESGGGLVQPGGSLRISCAASGFTFSAFGVSWVRQAPGK
    GLEWVSMIWGDGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC2_F8 542 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDLGVNWVRQAPGK
    GLEWVSTISDIGSTYYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARDWHNGRFDYWGQGTLVTVSS
    rHC1_F10 543 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSIIRGDGFTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_C12 544 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSIIRADGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_C11 545 EVQLVESGGGLVQPGGSLRLSCAASGFTFSHFGVNWVRQAPGK
    GLEWVSIIWGDGSTAYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_C4 546 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVEWVRQAPGK
    GLEWVSKIWADGSTDYADSLKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    H1 + H2_E12 547 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGVNWVRQAPGK
    GLEWVSLIWGDGTTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_C4 548 EVQLVESGGGLVQPGGSLRLSCAASGFTFSYFGVSWVRQAPGK
    GLEWVSMIWGDGSTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_F9 549 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRSDGSTDYADTLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    H1 + H2_B5 550 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFGVNWVRQAPGK
    GLEWVSIIWSDGSTDYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    S4-34 551 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMIWADGSTHYADTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    H1 + H2_C2 552 EVQLVESGGGLVQPGGSLRLSCAASGFTFSEFGVNWVRQAPGK
    GLEWVSMIWGNGATDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_F11 553 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFGVNWVRQAPGK
    GLEWVSMIWGDGTTAYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC2_E9 554 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRADGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_B2 555 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFGVNWVRQAPGK
    GLEWVSMIWGDGSTDYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_E9 556 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGVNWVRQAXGK
    GLEWVSMIWGDGSTDYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_A6 557 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMIGSDGFTDYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    H1 + H2_C8 558 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQTPGK
    GLEWVSMIRGDGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_C5 559 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFGVSWVRQAPGK
    GLEWVSQIWGDGSTDYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC2_D5 560 EVQLVESGGGLVQPGGSLRLSCAASGFTFSQLGVTWVRQAPGK
    GLEWVSTISDAGSTYYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARDWHHGRFAYWGQGTLVTVSS
    rHC1_C7 561 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRADGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARDWQHGPLGYWGQGTLVTVSS
    H1 + H2_C3 562 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYGVHWVRQAPGK
    GLEWVSMIWGDGSTDYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_G7 563 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRGDGTTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARDWQHGPIGYWGQGTLVTVSS
    rHC1_A5 564 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWADGYTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    H1 + H2_G9 565 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVEWVRQAPGK
    GLEWVSKIWGDGTTDYADTLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_E2 566 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIGGEGRTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_C9 567 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNLGVNWVRQAPGK
    GLEWVSMIWDVGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARDWHHGLFDYWGQGTLVTVSS
    rHC1_G6 568 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIMGDGYTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC1_C1 569 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRDDGATDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    rHC1_C2 570 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMISGDGYTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    H1 + H2_C1 571 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSIIRGDGSTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_B10 572 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGVNWVRQAPGX
    GLEWVSMIWADGSTDYASTLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_E3 573 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAFGVCWVRQAPGK
    GLEWVSMIWADGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_H4 574 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRSDGSTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARDWQHGPEGYWGQGTLVTVSS
    rHC2_A1 575 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIRGDGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_G11 576 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSLIRSDGSTHYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_D8 577 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIRGDGYTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_A3 578 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWADGSTHYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-31 579 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVQWVRQAPGK
    GLEWVSGIGADGSTAYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHSGLAYWGQGTLVTVSS
    rHC36 580 EVQLVESGGGLVQPGGSLILSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMIWADGSTHYASSLKGRFTISRDNFKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC2_G3 581 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIRGDGFTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_C10 582 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMIAADGSTAYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC14 583 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWADGSTHYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPAAYWGQGTLVTVSS
    rHC1_D4 584 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRGDGSTDYADTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC2_D11 585 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSIISGDGFTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC2_E11 586 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDWGVHWMRQAPGK
    GLEWVSTIWDDGSTYYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARHGHHGPFVYWGQGTLVTVSS
    H1 + H2_E7 587 EVQLVESGGGLVQPGGSLRLSCAASXFTFSNFGVNWVRQAPGK
    GLEWVSMIWGDGSTDYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_A8 588 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYGVNWVRQAPGK
    GLEWVSMIGDEGSTDYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARHWHHGAVDYWGQGTLVTVSS
    H1 + H2_B9 589 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGVNWVRQAPGK
    GLEWVSMIWADGSTHYADSLKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    S4-19 590 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVEWVRQAPGK
    GLEWVSGIWADGSTHYADTVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-74 591 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWADGSTHYADTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    rHC1_H2 592 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRGDGFTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC1_E3 593 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRADGYTSYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC34 594 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWADGSTHYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPSAYWGQGTLVTVSS
    H1 + H2_F2 595 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIRADGSTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_D9 596 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRADGTTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVGYWGQGTLVTVSS
    H1 + H2_E6 597 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGVHWVRQAPGK
    GLEWVSMIWADGSTVYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_F3 598 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIGSDGSTYYADSLKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_G11 599 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRGDGFTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLGYWGQGTLVTVSS
    H1 + H2_D3 600 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFGVNWVRQAPGK
    GLEWVSMIWGDGHTAYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_B12 601 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGVNWVRQAPGK
    GLEWVSMIWAHGATHYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_B11 602 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSLIRDDGSTDYASTLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_A8 603 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWGDGSTDYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    S4-24 604 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWADGSTHYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC1_F11 605 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMISADGYTDYADSLKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    H1 + H2_D10 606 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMIWADGSTHYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC2_D6 607 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIGADGYTDYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_G4 608 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAFGVSWVRQAPGK
    GLEWVSMIWADGSTDYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_D11 609 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSLIRGDGSTDYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_E9 610 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWADGTTYYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    rHC1_A12 611 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVQWVRQAPGK
    GLEWVSRISGDGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_A2 612 EVQLVESGGGLVQPGGSLRLSCAASGFSFSNFGVNWVRQAPGK
    GLEWVSMIWADGSTNYADTVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_B7 613 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYGVSWVRQAPGK
    GLEWVSIISADGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_H8 614 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRGDGSTDYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC1_F12 615 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIGADGYTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_E5 616 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSIIRGDGSTDYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_A11 617 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGVNWVRQAPGK
    GLEWVSMIWGSGATDYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_D6 618 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMISADGFTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC2_G10 619 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIAADGFTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_H3 620 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSLIAADGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    H1 + H2_F10 621 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSIIRGDGSTAYADTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_C7 622 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIWGDGNTGYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_A9 623 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRGDGSTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    H1 + H2_E5 624 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGVNWVRQAPGK
    GLEWVSMIWGDGSTEYADTLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC62 625 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMIWADGSTHYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVGYWGQGTLVTVSS
    H1 + H2_F4 626 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVYWVRQAPGK
    GLEWVSMIWDDGSTEYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC2_H8 627 EVQLVESGGGLVQPGGSLRLSCAASGFTFSQLGVTWVRQAPGK
    GLEWVSTISDAGSTYYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARDWHHGRFAYWGQGTLVTVSS
    rHC2_F4 628 EVQLVESGGGLVQPGGSLRLSCAASGFTFSGPGVNWVRQAPGK
    GLEWVSSIWDDGSTYYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCARHSHDGRFDYWGQGTLVTVSS
    S4-50 629 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVEWVRQAPGK
    GLEWVSGIWADGSTHYADTVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVGYWGQGTLVTVSS
    H1 + H2_F12 630 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGVNWVRQAPGK
    GLEWVSMIWGEGSTGYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_E6 631 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSIIRDDGFTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_F2 632 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIGGDGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    H1 + H2_G6 633 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFGVNWVRQAPGK
    GLEWVSMIWADGTTDYDDSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC2_F5 634 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSGISADGSTAYDSSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_D6 635 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGVSWVRQAPGK
    GLEWVSLIRGDGSTYYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_A9 636 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGVNWVRQAPGK
    GLEWVSMIWGDGSTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_A1 637 EVQLVESGGGLVQPGGSLRLSCAASGFTFSHFGVNWVRQAPGK
    GLEWVSMIWADGSTDYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC60 638 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMIWADGSTHYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPAAYWGQGTLVTVSS
    rHC1_C8 639 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVHWVRQAPGK
    GLEWVSMIAGDGSTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVGYWGQGTLVTVSS
    rHC44 640 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMIWADGSTHYADTLKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC1_G9 641 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSIIGADGATDYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLGYWGQGTLVTVSS
    H1 + H2_A6 642 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSGITGDGITAYASTLKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_G2 643 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMISGDGFTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_G7 644 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGVNWVRQAPGK
    GLEWVSNIWGDGSTDYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_E10 645 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIRADGSTDYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_E2 646 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRGDGSTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_A4 647 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYGVSWVRQAPGK
    GLEWVSMIWRDGSTDYADSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_H3 648 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVHWVRQAPGK
    GLEWVSMIWGDGSTHYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_G1 649 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVHWVRQAPGK
    GLEWVSGISADGSTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_E8 650 EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYGVNWVRQAPGK
    GLEWVSMIGGDGFTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_C9 651 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIRADGSTDYASSLKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_F7 652 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVQWVRQAPGK
    GLEWVSVISADGFTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_F6 653 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIGADGSTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVGYWGQGTLVTVSS
    rHC22 654 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWADGSTDYADTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC2_G5 655 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSLIRGDGYTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_C12 656 EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYGVSWVRQAPGK
    GLEWVSVIRADGVTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVGYWGQGTLVTVSS
    rHC3 657 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMIWADGSTHYASSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC1_F1 658 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVQWVRQAPGK
    GLEWVSRINGDGSTDYASTLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_E11 659 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIRSDGFTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_B8 660 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGVNWVRQAPGK
    GLEWVSMIWVDGSTDYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_G1 661 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGVNWVRQAPGK
    GLEWVSMIWGDGSTYYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_B3 662 EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYGVSWVRQAPGK
    GLEWVSMIRSDGFTDYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_D2 663 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMITGDGYTDYADTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    rHC1_E12 664 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSIIRADGLTDYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_B5 665 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSLIRSDGSTDYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_D11 666 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRADGSTDYADSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPLAYWGQGTLVTVSS
    H1 + H2_A7 667 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGVIWVRQAPGK
    GLEWVSMIGGDGSTYYDSSLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_G3 668 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVNWVRQAPGK
    GLEWVSMIGSDGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_D5 669 EVQLVESGGGLVQPGGSLRLSCAASGFTFSYYGVHWVRQAPGK
    GLEWVSGISGEGSTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_D1 670 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRGDGSTYYASSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWVKGTLVTVSS
    rHC1_E7 671 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSIIRGDGSTDYASSLKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    rHC1_E11 672 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIRADGTTDYASSVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    S4-55 673 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVTWVRQAPGK
    GLEWVSMIWADGSTDYASTVKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWQHGPVGYWGQGTLVTVSS
    H1 + H2_C10 674 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGVSWVRQAPGK
    GLEWVSMIRGDGSTYYADTLKGRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
    H1 + H2_G10 675 EVQLVESGGGLVQPGGSLRLSCAASGFTFSHFGVNWVRQAPGK
    GLEWVSMIWADGSTSYADSVKSRFTISRDNSKNTLYLQMNSLR
    AEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
  • Table 20 provides a list of amino acid sequences of VL regions of affinity matured fully human TNF antibodies derived from hMAK195 Amino acid residues of individual CDRs of each VH sequence are indicated in bold.
  • TABLE 20
    List of amino acid sequences of affinity matured
    hMAK195 VL variants
    SEQ
    ID
    Clone NO: VL
    S3_92 676 DIQMTQSPSSLSASVGDRVTITCRASQKVSSAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYHTPYTFGQGTKLEIK
    S3_79 677 DIQMTQSPSSLSASVGDRVTITCKASQAVSTEVAWYQQK
    PGKAPKLLIYCASTRQTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQSYSAPYTFGQGTKLEIK
    S3_68 678 DIQMTQSPSSLSASVGDRVTITCRASQVVSSAVAWYQQK
    PGKAPKLLIYWASKRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
    S3_60 679 DIQMTQSPSSLSASVGDRVTITCRASQAVSSAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
    S4-63 680 DIQMTQSPSSLSASVGDRVTITCKASQKVSSALAWYQQK
    PGKAPKLLIYWASALHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRPPFTFGQGTKLEIK
    S3_5 681 DIQMTQSPSSLSASVGDRVTITCRASQGVSSAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYTTPFTFGQGTKLEIK
    S3_44 682 DIQMTQSPSSLSASVGDRVTITCRASQGVSRALAWYQQK
    PGKAPKLLIYWASTLHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRAPFTFGQGTKLEIK
    S3_53 683 DIQMTQSPSSLSASVGDRVTITCRASQAVSSAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYHTPFTFGQGTKLEIK
    S3_91 684 DIQMTQSPSSLSASVGDRVTITCKASQGVSSALAWYQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPFTFGQGTKLEIK
    S3_59 685 DIQMTQSPSSLSASVGDRVTITCKASQGVSSALAWYQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPYTFGQGTKLEIK
    S3_47 686 DIQMTQSPSSLSASVGDRVTITCKASQWVSSAVAWYQQK
    PGKAPKLLIYWASTRQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRIPFTFGQGTKLEIK
    S3_70 687 DIQMTQSPSSLSASVGDRVTITCKASQAVSSALAWYQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPYTFGQGTKLEIK
    S3_56 688 DIQMTQSPSSLSASVGDRVTITCKASQRVSSAVAWYQQK
    PGKAPKLLIYWASTLHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPYTFGQGTKLEIK
    S3_37 689 DIQMTQSPSSLSASVGDRVTITCKASQGVSSAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYNTPFTFGQGTKLEIK
    S3_36 690 DIQMTQSPSSLSASVGDRVTITCKASQKVSSAVAWYQQK
    PGKAPKLLIYWASARHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
    S3_67 691 DIQMTQSPSSLSASVGDRVTITCKASQTVXRAVAWYQQK
    PGKAPKLLIYWASTRQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQSYSTPFTFGQGTKLEIK
    S3_40 692 DIQMTQSPSSLSASVGDRVTITCRASQRVSSAVAWSQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYTTPYTFGQGTKLEIK
    S3_73 693 DIQMTQSPSSLSASVGDRVTITCKASQAVSSAVAWYQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
    S4-50 694 DIQMTQSPSSLSASVGDRVTITCKASQLVSSAVAWYQQK
    PGKAPKLLIYWASALHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSSPYTFGQGTKLEIK
    S4-6 695 DIQMTQSPSSLSASVGDRVTITCKASQLVSSAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
    S3_19 696 DIQMTQSPSSLSASVGDRVTITCKASQKVSSAVAWYQQK
    PGKAPKLLIYWASARHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRSPFTFGQGTKLEIK
    S3_83 697 DIQMTQSPSSLSASVGDRVTITCRASQAVSTALAWYQQK
    PGKAPKLLIYSASTLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRSPFTFGQGTKLEIK
    S3_78 698 DIQMTQSPSSLSASVGDRVTITCKASQYVGGAVAWYQQK
    PGKAPKLLIYQASTLQTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHISKPFTFGQGTKLEIK
    S4-19 699 DIQMTQSPSSLSASVGDRVTITCKASQLVSSAVAWYQQK
    PGKAPKLLIYWASTLHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPFTFGQGTKLEIK
    S3_58 700 DIQMTQSPSSLSASVGDRVTITCKASQSVNGALAWYQQK
    PGKAPKLLIYRASTRQTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSIPFTFGQGTKLEIK
    S4-31 701 DIQMTQSPSSLSASVGDRVTITCRASQGVSSALAWYQQK
    PGKAPKLLIYWASALHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSAPFTFGQGTKLEIK
    S3_31 702 DIQMTQSPSSLSASVGDRVTITCKASQAVSSSVAWYQQK
    PGKAPKLLIYGASTLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYNEPYTFGQGTKLEIK
    S3_13 703 DIQMTQSPSSLSASVGDRVTITCKASQKVSSAVAWYQQK
    PGKAPKLLIYWASARHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPYTFGQGTKLEIK
    S4-40 704 DIQMTQSPSSLSASVGDRVTITCRASQLVSSAVAWYQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPFSFGQGTKLEIK
    S3_26 705 DIQMTQSPSSLSASVGDRVTITCRASQAVSSAVAWYQQK
    PGKAPKLLIYWASKRQTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYISPYTFGQGTKLEIK
    S3_33 706 DIQMTQSPSSLSASVGDRVTITCKASQGVRSALAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQSYSAPYTFGQGTKLEIK
    S3_28 707 DIQMTQSPSSLSASVGDRVTITCKASQTVSNAVAWYQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
    S4-74 708 DIQMTQSPSSLSASVGDRVTITCRASQLVSSAVAWYQQK
    PGKAPKLLIYWASARHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPFTFGQGTKLEIK
    S3_84 709 DIQMTQSPSSLSASVGDRVTITCKASQPVRSAVAWYQQK
    PGKAPKLLIYSASTRQTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQSYSIPFTFGQGTKLEIK
    S4-54 710 DIQMTQSPSSLSASVGDRVTITCRASQLVSSAVAWYQQK
    PGKAPKLLIYWASARHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYKTPFSFGQGTKLEIK
    S3_23 711 DIQMTQSPSSLSASVGDRVTITCRASQAVSSAVAWYQQK
    PGKAPKLLIYWASSRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
    S3_55 712 DIQMTQSPSSLSASVGDRVTITCKASQTVGRAVAWYQQK
    PGKAPKLLIYWASTRQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQSYSTPFTFGQGTKLEIK
    S4-34 713 DIQMTQSPSSLSASVGDRVTITCRASQLVSSAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPFTFGQGTKLEIK
    S3_76 714 DIQMTQSPSSLSASVGDRVTITCRASQKVSNAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYNSPFTFGQGTKLEIK
    S4-12 715 DIQMTQSPSSLSASVGDRVTITCRASQLVSSAVAWYQQK
    PGKAPKLLIYWASARHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYKTPFTFGQGTKLEIK
    S3_86 716 DIQMTQSPSSLSASVGDRVTITCRASQRVSSAVAWYQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYTTPYTFGQGTKLEIK
    S3_61 717 DIQMTQSPSSLSASVGDRVTITCKASQRVSSAVAWYQQK
    PGKAPKLLIYWASNRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
    S3_18 718 DIQMTQSPSSLSASVGDRVTITCKASQLVSSALAWYQQK
    PGKAPKLLIYWASTRQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPFTFGQGTKLEIK
    S3_72 719 DIQMTQSPSSLSASVGDRVTITCKASQLVSSALAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRNPFTFGQGTKLEIK
    S3_41 720 DIQMTQSPSSLSASVGDRVTITCKASQAVSSALAWYQQK
    PXKAPKLLIYWASSRQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPFTFGQGTKLEIK
    S4-24 721 DIQMTQSPSSLSASVGDRVTITCRASQLVSSAVAWYQQK
    PGKAPKLLIYWASTLHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPFTFGQGTKLEIK
    S4-17 722 DIQMTQSPSSLSASVGDRVTITCRASQLVSSAVAWYQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPFTFGQGTKLEIK
    S3_90 723 DIQMTQSPSSLSASVGDRVTITCKASQPVSGAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRASYTFGQGTKLEIK
    S3_87 724 DIQMTQSPSSLSASVGDRVTITCRASQKVSSAVAWYQQK
    PGKAPKLLIYWASARHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPYTFGQGTKLEIK
    S3_66 725 DIQMTQSPSSLSASVGDRVTITCRASQRVSSAVAWYQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYTTPYTFGQGTKLEIK
    S4-18 726 DIQMTQSPSSLSASVGDRVTITCRASQLVSSAVAWYQQK
    PGKAPKLLIYWASTLHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
    S3_4 727 DIQMTQSPSSLSASVGDRVTITCRASQAVSSAVAWYQQK
    PGKAPKLLIYWASARHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSSPYTFGQGTKLEIK
    S3_64 728 DIQMTQSPSSLSASVGDRVTITCKASQPVSSAVAWYQQK
    PGKAPKLLIYWASTLHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPFTFGQGTKLEIK
    S3_62 729 DIQMTQSPSSLSASVGDRVTITCRASQLVSSAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPYTFGQGTNLEIK
    S3_29 730 DIQMTQSPSSLSASVGDIVTITCKASQLVSSAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRTPYTFGQGTKLEIK
    S3_65 731 DIQMTQSPSSLSASVGDRVTITCKASQLVSSAVAWYQQK
    PGKAPKLLIYWASMRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSSPFTFGQGTKLEIK
    S3_81 732 DIQMTQSPSSLSASVGDRVTITCKASQTVSSAVAWYQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYRAPYTFGQGTKLEIK
    S3_39 733 DIQMTQSPSSLSASVGDRVTITCKASQRVSSALAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
    S3_49 734 DIQMTQSPSSLSASVGDRVTITCRASQLVSNAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSSPFTFGQGTKLEIK
    S3_85 735 DIQMTQSPSSLSASVGDRVTITCRASQLVSSAVAWYQQK
    PGKAPKLLIYWASARHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
    S3_82 736 DIQMTQSPSSLSASVGDRVTITCKASQLVSSAVAWYQQK
    PGKAPKLLIYWASTRHSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYTTPFTFGQGTKLEIK
    S3_93 737 DIQMTQSPSSLSASVGDRVTITCKASQRVSSAVAWYQQK
    PGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQHYSTPFTFGQGTKLEIK
  • TABLE 21
    Amino acid residues observed in affinity matured hMAK-195.
    hMAK195 Heavy chain variable region (SEQ ID NO: 1075)
    hMAK195VH EVQLVESGGGLVQPGGSLRLSCAASGFTFS DYGVN WVRQAPGKGLEWVS MIWGDGSTD
                                  NFS T              I RAG T A
                                  HLN S              V GSE F H
                                  YS  H              L SDA A V
                                  IR  Q              R AEV Y S
                                      Y              K LVG W N
                                                     S  NY   G
    YDSTLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREWHHGPVAYWGQGTLVTVSS
     ADSV G                                HSQQRTLDS
                                           QLRPASGVF
                                           LCLLVQDGC
                                           YRYNWAETN
                                           DFPYEKW P
                                           NDARS R I
                                           TYVTP P H
                                           PPDDI A
                                           AICA  I
                                           SG C
                                           R
    hMAK195 Light chain variable region (SEQ ID NO: 1076)
    hMAK195VL DIQMTQSPSSLSASVGDRVTITC KASQAVSSAVA WYQQKPGKAPKLLIY WASTRHT G
                           R   S RRPL                S  SLQS
                               V TNT                 R  I T
                               G IGG                 L  L A
                               D NCV                 C  K E
                               T CTS                 Q  A F
                               P KIR                 G  R
    VPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYSTPFT FGQGTKLEIK
                                     SNRSTY
                                      FGPR
                                      DTML
                                      GIIQ
                                      HCAA
                                         S
  • The tables below provide a list of humanized MAK-195 antibodies that were converted into IgG proteins for characterization.
  • TABLE 22
    VH sequences of IgG converted clones
    Protein Sequence
    region SEQ ID NO: 123456789012345678901234567890
    A8 738 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVNWVRQAPGKGLEWVSMIAADGFTDYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWHHGPVAYWGQGTLVTVSS
    A8 CDR-H1 Residues 31-35 NYGVN
    VH of SEQ ID
    NO.: 738
    A8 CDR-H2 Residues 50-65 MIAADGFTDYASSVKG
    VH of SEQ ID
    NO.: 738
    A8 CDR-H3 Residues 98-106 EWHHGPVAY
    VH of SEQ ID
    NO.: 738
    B5 739 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVSWVRQAPGKGLEWVSLIRGDGSTDYA
    SSLKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWHHGPVAYWGQGTLVTVSS
    B5 CDR-H1 Residues 31-35 NYGVS
    VH of SEQ ID
    NO.: 739
    B5 CDR-H2 Residues 50-65 LIRGDGSTDYASSLKG
    VH of SEQ ID
    NO.: 739
    B5 CDR-H3 Residues 98-106 EWHHGPVAY
    VH of SEQ ID
    NO.: 739
    rHC44 740 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVSWVRQAPGKGLEWVSMIWADGSTHYA
    DTLKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC44 CDR-H1 Residues 31-35 NYGVS
    VH of SEQ ID
    NO.: 740
    rHC44 CDR-H2 Residues 50-65 MIWADGSTHYADTLKS
    VH of SEQ ID
    NO.: 740
    rHC44 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 740
    rHC22 741 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTDYA
    DTVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC22 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 741
    rHC22 CDR-H2 Residues 50-65 MIWADGSTDYADTVKG
    VH of SEQ ID
    NO.: 741
    rHC22 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 741
    rHC81 742 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    DSVKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPLAYWGQGTLVTVSS
    rHC81 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 742
    rHC81 CDR-H2 Residues 50-65 MIWADGSTHYADSVKS
    VH of SEQ ID
    NO.: 742
    rHC81 CDR-H3 Residues 98-106 EWQHGPLAY
    VH of SEQ ID
    NO.: 742
    rHC18 743 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWSDGSTDYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC18 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 743
    rHC18 CDR-H2 Residues 50-65 MIWSDGSTDYASSVKG
    VH of SEQ ID
    NO.: 743
    rHC18 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 743
    rHC14 744 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSLKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPAAYWGQGTLVTVSS
    rHC14 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 744
    rHC14 CDR-H2 Residues 50-65 MIWADGSTHYASSLKG
    VH of SEQ ID
    NO.: 744
    rHC14 CDR-H3 Residues 98-106 EWQHGPAAY
    VH of SEQ ID
    NO.: 744
    rHC3 745 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVSWVRQAPGKGLEWVSMIWADGSTHYA
    SSLKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC3 CDR-H1 Residues 31-35 NYGVS
    VH of SEQ ID
    NO.: 745
    rHC3 CDR-H2 Residues 50-65 MIWADGSTHYASSLKG
    VH of SEQ ID
    NO.: 745
    rHC3 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 745
    rHC19 746 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPAAYWGQGTLVTVSS
    rHC19 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 746
    rHC19 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 746
    rHC19 CDR-H3 Residues 98-106 EWQHGPAAY
    VH of SEQ ID
    NO.: 746
    rHC34 747 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPSAYWGQGTLVTVSS
    rHC34 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 747
    rHC34 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 747
    rHC34 CDR-H3 Residues 98-106 EWQHGPSAY
    VH of SEQ ID
    NO.: 747
    rHC83 748 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    rHC83 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 748
    rHC83 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 748
    rHC83 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 748
    S4-19 749 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVEWVRQAPGKGLEWVSGIWADGSTHYA
    DTVKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-19 CDR-H1 Residues 31-35 NYGVE
    VH of SEQ ID
    NO.: 749
    S4-19 CDR-H2 Residues 50-65 GIWADGSTHYADTVKS
    VH of SEQ ID
    NO.: 749
    S4-19 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 749
    S4-50 750 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVEWVRQAPGKGLEWVSGIWADGSTHYA
    DTVKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVGYWGQGTLVTVSS
    S4-50 CDR-H1 Residues 31-35 NYGVE
    VH of SEQ ID
    NO.: 750
    S4-50 CDR-H2 Residues 50-65 GIWADGSTHYADTVKS
    VH of SEQ ID
    NO.: 750
    S4-50 CDR-H3 Residues 98-106 EWQHGPVGY
    VH of SEQ ID
    NO.: 750
    S4-63 751 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVEWVRQAPGKGLEWVSGIWADGSTHYA
    DTVKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVGYWGQGTLVTVSS
    S4-63 CDR-H1 Residues 31-35 NYGVE
    VH of SEQ ID
    NO.: 751
    S4-63 CDR-H2 Residues 50-65 GIWADGSTHYADTVKS
    VH of SEQ ID
    NO.: 751
    S4-63 CDR-H3 Residues 98-106 EWQHGPVGY
    VH of SEQ ID
    NO.: 751
    S4-55 752 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTDYA
    STVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVGYWGQGTLVTVSS
    S4-55 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 752
    S4-55 CDR-H2 Residues 50-65 MIWADGSTDYASTVKG
    VH of SEQ ID
    NO.: 752
    S4-55 CDR-H3 Residues 98-106 EWQHGPVGY
    VH of SEQ ID
    NO.: 752
    S4-6 753 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-6 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 753
    S4-6 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 753
    S4-6 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 753
    S4-18 754 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    DSVKSRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPLAYWGQGTLVTVSS
    S4-18 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 754
    S4-18 CDR-H2 Residues 50-65 MIWADGSTHYADSVKS
    VH of SEQ ID
    NO.: 754
    S4-18 CDR-H3 Residues 98-106 EWQHGPLAY
    VH of SEQ ID
    NO.: 754
    S4-31 755 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVQWVRQAPGKGLEWVSGIGADGSTAYA
    SSLKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHSGLAYWGQGTLVTVSS
    S4-31 CDR-H1 Residues 31-35 NYGVQ
    VH of SEQ ID
    NO.: 755
    S4-31 CDR-H2 Residues 50-65 GIGADGSTAYASSLKG
    VH of SEQ ID
    NO.: 755
    S4-31 CDR-H3 Residues 98-106 EWQHSGLAY
    VH of SEQ ID
    NO.: 755
    S4-34 756 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVSWVRQAPGKGLEWVSMIWADGSTHYA
    DTVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPLAYWGQGTLVTVSS
    S4-34 CDR-H1 Residues 31-35 NYGVS
    VH of SEQ ID
    NO.: 756
    S4-34 CDR-H2 Residues 50-65 MIWADGSTHYADTVKG
    VH of SEQ ID
    NO.: 756
    S4-34 CDR-H3 Residues 98-106 EWQHGPLAY
    VH of SEQ ID
    NO.: 756
    S4-74 757 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    DTVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPLAYWGQGTLVTVSS
    S4-74 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 757
    S4-74 CDR-H2 Residues 50-65 MIWADGSTHYADTVKG
    VH of SEQ ID
    NO.: 757
    S4-74 CDR-H3 Residues 98-106 EWQHGPLAY
    VH of SEQ ID
    NO.: 757
    S4-12 758 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-12 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 758
    S4-12 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 758
    S4-12 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 758
    S4-54 759 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-54 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 759
    S4-54 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 759
    S4-54 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 759
    S4-17 760 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-17 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 760
    S4-17 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 760
    S4-17 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 760
    S4-40 761 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-40 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 761
    S4-40 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 761
    S4-40 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 761
    S4-24 762 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
    VH NYGVTWVRQAPGKGLEWVSMIWADGSTHYA
    SSVKGRFTISRDNSKNTLYLQMNSLRAEDT
    AVYYCAREWQHGPVAYWGQGTLVTVSS
    S4-24 CDR-H1 Residues 31-35 NYGVT
    VH of SEQ ID
    NO.: 762
    S4-24 CDR-H2 Residues 50-65 MIWADGSTHYASSVKG
    VH of SEQ ID
    NO.: 762
    S4-24 CDR-H3 Residues 98-106 EWQHGPVAY
    VH of SEQ ID
    NO.: 762
  • TABLE 23
    VL sequences of IgG converted clones
    Protein Sequence
    region SEQ ID NO: 123456789012345678901234567890
    hMAK195 763 DIQMTQSPSSLSASVGDRVTITCKASQAVS
    VL.1 SAVAWYQQKPGKAPKLLIYWASTRHTGVPS
    VL RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYSTPFTFGQGTKLEIKR
    hMAK195 CDR-L1 Residues 24-34 KASQAVSSAVA
    VL.1 of SEQ ID
    VL NO.: 763
    hMAK195 CDR-L2 Residues 50-56 WASTRHT
    VL.1 of SEQ ID
    VL NO.: 763
    hMAK195 CDR-L3 Residues 89-97 QQHYSTPFT
    VL.1 of SEQ ID
    VL NO.: 763
    S4-24 764 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTLHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-24 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 764
    S4-24 CDR-L2 Residues 50-56 WASTLHT
    VL of SEQ ID
    NO.: 764
    S4-24 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 764
    S4-40 765 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTRHSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFSFGQGTKLEIKR
    S4-40 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 765
    S4-40 CDR-L2 Residues 50-56 WASTRHS
    VL of SEQ ID
    NO.: 765
    S4-40 CDR-L3 Residues 89-97 QQHYRTPFS
    VL of SEQ ID
    NO.: 765
    S4-17 766 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTRHSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-17 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 766
    S4-17 CDR-L2 Residues 50-56 WASTRHS
    VL of SEQ ID
    NO.: 766
    S4-17 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 766
    S4-54 767 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASARHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYKTPFSFGQGTKLEIKR
    S4-54 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 767
    S4-54 CDR-L2 Residues 50-56 WASARHT
    VL of SEQ ID
    NO.: 767
    S4-54 CDR-L3 Residues 89-97 QQHYKTPFS
    VL of SEQ ID
    NO.: 767
    S4-12 768 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASARHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYKTPFTFGQGTKLEIKR
    S4-12 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 768
    S4-12 CDR-L2 Residues 50-56 WASARHT
    VL of SEQ ID
    NO.: 768
    S4-12 CDR-L3 Residues 89-97 QQHYKTPFT
    VL of SEQ ID
    NO.: 768
    S4-74 769 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASARHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-74 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 769
    S4-74 CDR-L2 Residues 50-56 WASARHT
    VL of SEQ ID
    NO.: 769
    S4-74 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 769
    S4-34 770 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTRHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-34 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 770
    S4-34 CDR-L2 Residues 50-56 WASTRHT
    VL of SEQ ID
    NO.: 770
    S4-34 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 770
    S4-31 771 DIQMTQSPSSLSASVGDRVTITCRASQGVS
    VL SALAWYQQKPGKAPKLLIYWASALHSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYSAPFTFGQGTKLEIKR
    S4-31 CDR-L1 Residues 24-34 RASQGVSSALA
    VL of SEQ ID
    NO.: 771
    S4-31 CDR-L2 Residues 50-56 WASALHS
    VL of SEQ ID
    NO.: 771
    S4-31 CDR-L3 Residues 89-97 QQHYSAPFT
    VL of SEQ ID
    NO.: 771
    S4-18 772 DIQMTQSPSSLSASVGDRVTITCRASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTLHSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYSTPFTFGQGTKLEIKR
    S4-18 CDR-L1 Residues 24-34 RASQLVSSAVA
    VL of SEQ ID
    NO.: 772
    S4-18 CDR-L2 Residues 50-56 WASTLHS
    VL of SEQ ID
    NO.: 772
    S4-18 CDR-L3 Residues 89-97 QQHYSTPFT
    VL of SEQ ID
    NO.: 772
    S4-6 773 DIQMTQSPSSLSASVGDRVTITCKASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTRHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYSTPFTFGQGTKLEIKR
    S4-6 CDR-L1 Residues 24-34 KASQLVSSAVA
    VL of SEQ ID
    NO.: 773
    S4-6 CDR-L2 Residues 50-56 WASTRHT
    VL of SEQ ID
    NO.: 773
    S4-6 CDR-L3 Residues 89-97 QQHYSTPFT
    VL of SEQ ID
    NO.: 773
    S4-55 774 DIQMTQSPSSLSASVGDRVTITCKASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTLHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-55 CDR-L1 Residues 24-34 KASQLVSSAVA
    VL of SEQ ID
    NO.: 774
    S4-55 CDR-L2 Residues 50-56 WASTLHT
    VL of SEQ ID
    NO.: 774
    S4-55 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 774
    S4-63 775 DIQMTQSPSSLSASVGDRVTITCKASQKVS
    VL SALAWYQQKPGKAPKLLIYWASALHSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRPPFTFGQGTKLEIKR
    S4-63 CDR-L1 Residues 24-34 KASQKVSSALA
    VL of SEQ ID
    NO.: 775
    S4-63 CDR-L2 Residues 50-56 WASALHS
    VL of SEQ ID
    NO.: 775
    S4-63 CDR-L3 Residues 89-97 QQHYRPPFT
    VL of SEQ ID
    NO.: 775
    S4-50 776 DIQMTQSPSSLSASVGDRVTITCKASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASALHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYSSPYTFGQGTKLEIKR
    S4-50 CDR-L1 Residues 24-34 KASQLVSSAVA
    VL of SEQ ID
    NO.: 776
    S4-50 CDR-L2 Residues 50-56 WASALHT
    VL of SEQ ID
    NO.: 776
    S4-50 CDR-L3 Residues 89-97 QQHYSSPYT
    VL of SEQ ID
    NO.: 776
    S4-19 777 DIQMTQSPSSLSASVGDRVTITCKASQLVS
    VL SAVAWYQQKPGKAPKLLIYWASTLHTGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    HYRTPFTFGQGTKLEIKR
    S4-19 CDR-L1 Residues 24-34 KASQLVSSAVA
    VL of SEQ ID
    NO.: 777
    S4-19 CDR-L2 Residues 50-56 WASTLHT
    VL of SEQ ID
    NO.: 777
    S4-19 CDR-L3 Residues 89-97 QQHYRTPFT
    VL of SEQ ID
    NO.: 777
  • TABLE 24
    Heavy and light chain pairs of hMAK195 affinity matured clones
    Clone name HC LC Protein name
    A8 hMAK195-A8 hMAK195 VL.1 hMAK195-AM11
    B5 hMAK195-B5 hMAK195 VL.1 hMAK195-AM13
    rHC3 hMAK195 rHC3 hMAK195 VL.1 hMAK195-AM14
    rHC18 hMAK195 rHC18 hMAK195 VL.1 hMAK195-AM15
    rHC19 hMAK195 rHC19 hMAK195 VL.1 hMAK195-AM16
    rHC22 hMAK195 rHC22 hMAK195 VL.1 hMAK195-AM17
    rHC34 hMAK195 rHC34 hMAK195 VL.1 hMAK195-AM18
    rHC60 hMAK195 rHC60 hMAK195 VL.1 hMAK195-AM19
    S4-6 hMAK195 S4-6 hMAK195 S4-6 hMAK195-AM20
    S4-12 hMAK195 S4-12 hMAK195 S4-12 hMAK195-AM21
    S4-17 hMAK195 S4-17 hMAK195 S4-17 hMAK195-AM22
    S4-18 hMAK195 S4-18 hMAK195 S4-18 hMAK195-AM23
    S4-19 hMAK195 S4-19 hMAK195 S4-19 hMAK195-AM24
    S4-24 hMAK195 S4-24 hMAK195 S4-24 hMAK195-AM25
    S4-34 hMAK195 S4-34 hMAK195 S4-34 hMAK195-AM26
    • 2.1 TNF Enzyme-Linked Immunosorbent Assay Result
  • TABLE 25
    IgG Name EC50 in hTNFa ELISA (nM)
    hMAK195-AM11 0.2
    hMAK195-AM13 0.2
    hMAK195-AM14 0.051
    hMAK195-AM15 0.052
    hMAK195-AM16 0.056
    hMAK195-AM17 0.056
    hMAK195-AM18 0.052
    hMAK195-AM19 0.057
    hMAK195-AM20 0.043
    hMAK195-AM21 0.042
    hMAK195-AM22 0.052
    hMAK195-AM23 0.055
    hMAK195-AM24 0.053
    hMAK195-AM25 0.052
    hMAK195-AM26 0.061
    • 2.2 TNF Neutralization Potency of TNF Antibodies by L929 Bioassay
  • TABLE 26
    hu TNF neutralization IC50 rhesus TNF
    IgG Name (nM) neutralization IC50 (nM)
    hMAK195-AM11 0.259 >25
    hMAK195-AM13 1.218 4.64
    hMAK195-AM14 0.0401 4.61
    hMAK195-AM15 0.036 >150
    hMAK195-AM16 0.0105 0.803
    hMAK195-AM17 0.0031 >25
    hMAK195-AM18 0.0145 0.4412
    hMAK195-AM19 0.0126 1.206
    hMAK195-AM20 0.0037 0.596
    hMAK195-AM21 0.009 0.09
    hMAK195-AM22 0.00345 0.2705
    hMAK195-AM23 0.0468 2.627
    hMAK195-AM24 0.015 0.557
    hMAK195-AM25 0.0114 0.262
    hMAK195-AM26 0.0061 0.2495
    • Example 3 Affinity Maturation of a Humanized Anti-Human TNF Antibody hMAK-199
  • The mouse anti-human TNF antibody MAK-199 was humanized and affinity-matured to generate a panel of humanized MAK195 variants that have improved affinity and binding kinetics against both human and cyno TNF. Several libraries were made according to specifications below:
  • Three HC libraries were made after the V2I back-mutation was first introduced and confirmed that it did not impact scFv affinity to TNF.
    H1+H2 (DDK) library:
  • Limited mutagenesis at 7 residues (T30, N31, N35, T52a, T54, E56, T58)
  • Germline toggle: M34I and F63L
  • H1+H2 (QKQ) library:
  • Limited mutagenesis at 7 residues (T30, N31, N35, T52a, T54, E56, T58)
  • Germline toggle: M34I and F63L
  • Germline back-mutations: D61Q, D62K, K64Q, F67V, F69M, L71T
  • H3 library:
  • Limited mutagenesis at 12 residues 95-100, 100a-100f
  • Germline toggle: F91Y
  • LC library: library
  • Limited mutagenesis at 11 residues 28, 30-32, 50, 53, 91-94, 96
  • Germline toggles: T51A, Y71F, F87Y, and T43A/V44P (these two co-evolve)
  • Recombined libraries:
    VH libraries will be recombined with and without VL library after library diversity is reduced after at least 3 rounds of selection.
  • All four libraries were selected separately for the ability to bind human or cynomolgus monkey TNF in the presence of decreasing concentrations of biotinylated human or cynomolgus monkey TNF antigens. All mutated CDR sequences recovered from library selections were recombined into additional libraries and the recombined libraries were subjected to more stringent selection conditions before individual antibodies are identified.
  • Table 27 provides a list of amino acid sequences of VH of the hMAK-199 antibody which were subjected to the affinity maturation selection protocol Amino acid residues of individual CDRs of each VH sequence are indicated in bold.
  • TABLE 27
    List of amino acid sequences of affinity matured
    hMAK199 VH variants
    Clone SEQ ID NO: VH
    J644M2S1-10VH 778 EVQLVQSGAEVKKPGASVKVSCKASGYTFNDYGITWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-11VH 779 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-12VH 780 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQ
    APGQGLEWMGWINTYTGEPHYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-13VH 781 EVQLVQSGAEVKKPGASVKVSCKASGYTFDNYGIQWVRQ
    APGQGLEWMGWINTYTGAPSYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-14VH 782 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGINWVRQ
    APGQGLEWMGWINTYTGKPSYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-15VH 783 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGMNWVRQ
    APGQGLEWMGWINTYTGESTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-16VH 784 EVQLVQSGAEVKKPGASVKVSCKASGYTFKNYGMTWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-17VH 785 EVQLVQSGAEVKKPGASVKVSCKASGYAFTDYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-18VH 786 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGEPAYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-1VH 787 EVQLVQSGAEVKKPGASVKVSCKASGYTFRNYGINWVRQ
    APGQGLEWMGWINTYTGQPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-22VH 788 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGINWVRQ
    APGQGLEWMGWINTYTGEPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-23VH 789 EVQLVQSGAEVKKPGASVKVSCKASGYTFKNYGIIWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-24VH 790 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGINWVRQ
    APGQGLEWMGWINTYTGVPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-25VH 791 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGKPSYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-27VH 792 EVQLVQSGAEVKKPGASVKVSCKASGYTFKNYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-28VH 793 EVQLVQSGAEVKKPGASVKVSCKASGYTFRNYGINWVRQ
    APGQGLEWMGWINTYTGKPSYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-2VH 794 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGIXWVRQ
    APGQGLEWMGWINTYXGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-31VH 795 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGEPHYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-33VH 796 EVQLVQSGAEVKKPGASVKVSCKASGYTFTHYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-34VH 797 EVQLVQSGAEVKKPGASVKVSCKASGYTFTHYGINWVRQ
    APGQGLEWMGWINTYTGQPTYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-35VH 798 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGITWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-36VH 799 EVQLVQSGAEVKKPGASVKVSCKASGYTFGNYGINWVRQ
    APGQGLEWMGWINTYTGKPSYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-37VH 800 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGINWVRQ
    APGQGLEWMGWINTYTGRPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-38VH 801 EVQLVQSGAEVKKPGASVKVSCKASGYTFKNYGINWVRQ
    APGQGLEWMGWINTYTGEPHYAQGFTGRVTMTTDTSTST
    AYIELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-3VH 802 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGEPSYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-40VH 803 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-41VH 804 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGIGWVRQ
    APGQGLEWMGWINTYTGKPSYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-43VH 805 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGVPSYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-44VH 806 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGIAWVRQ
    APGQGLEWMGWINTYTGVPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-45VH 807 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGVPHYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-46VH 808 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGIXWVRQ
    APGQGLEWMGWINTYTGEPXYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-47VH 809 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGVPTYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-48VH 810 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGQPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-4VH 811 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGITWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-50VH 812 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGVPQYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-51VH 813 EVQLVQSGAEVKKPGASVKVSCKASGYTFQNYGINWVRQ
    APGQGLEWMGWINTYTGVPTYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-53VH 814 EVQLVQSGAEVKKPGASVKVSCKASGYTFTQYGINWVRQ
    APGQGLEWMGWINTYTGDPHYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-54VH 815 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGINWVRQ
    APGQGLEWMGWINTYTGLPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-55VH 816 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYNGKPMYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-56VH 817 EVQLVQSGAEVKKPGASVKVSCKASGYTFRNYGITWVRQ
    APGQGLEWMGWINTYTGEPAYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-59VH 818 EVQLVQSGAEVKKPGASVKVSCKASGYTFNHYGINWVRQ
    APGQGLEWMGWINTYTGRPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-5VH 819 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-60VH 820 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-64VH 821 EVQLVQSGAEVKKPGASVKVSCKASGYTFDNYGINWVRQ
    APGQGLEWMGWINTYTGVPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-65VH 822 EVQLVQSGAEVKKPGASVKVSCKASGYTFNDYGIIWVRQ
    APGQGLEWMGWINTYTGKPSYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-66VH 823 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-67VH 824 EVQLVQSGAEVKKPGASVKVSCKASGYTFANYGMNWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-68VH 825 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGEPSYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-6VH 826 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGVPTYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-71VH 827 EVQLVQSGAEVKKPGASVKVSCKASGYTFDHYGMNWVRQ
    APGQGLEWMGWINTYTGKPTYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-72VH 828 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGIGWVRQ
    APGQGLEWMGWINTYTGKPSYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-73VH 829 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-74VH 830 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGMNWVRQ
    APGQGLEWMGWINTYTGKPTYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-75VH 831 EVQLVQSGAEVKKPGASVKVSCKASGYTFDNYGMNWVRQ
    APGQGLEWMGWINTYTGVPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-76VH 832 EVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-77VH 833 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGITWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-79VH 834 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYNGQPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-7VH 835 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGIIWVRQ
    APGQGLEWMGWINTYTGEPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-81VH 836 EVQLVQSGAEVKKPGASVKVSCKASGYTFANYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-82VH 837 EVQLVQSGAEVKKPGASVKVSCKASGYTFSDYGIQWVRQ
    APGQGLEWMGWINTYTGRPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-83VH 838 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGISWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-84VH 839 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGIQWVRQ
    APGQGLEWMGWINTYTGVPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-85VH 840 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGVPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-87VH 841 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYSGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-88VH 842 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGINWVRQ
    APGQGLEWMGWINTYTGQPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-8VH 843 EVQLVQSGAEVKKPGASVKVSCKASGYTFPNYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-90VH 844 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGKTNYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-91VH 845 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGEPNYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-92VH 846 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGITWVRQ
    APGQGLEWMGWINTYTGEPHYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-93VH 847 EVQLVQSGAEVKKPGASVKVSCKASGYTFKNYGINWVRQ
    APGQGLEWMGWINTYTGQPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-94VH 848 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGIPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-95VH 849 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-96VH 850 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGINWVRQ
    APGQGLEWMGWINTYSGVPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J644M2S1-9VH 851 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2-11VH 852 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFWRTVVGTDNAMDYWGQG
    TTVTVSS
    J647M2-12VH 853 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKYSTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2-13VH 854 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDSAMDYWGQG
    TTVTVSS
    J647M2-15VH 855 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFMTTMAVTDFAMDYWGQG
    TTVTVSS
    J647M2-16VH 856 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLLTTVVATDNAMDYWGQG
    TTVTVSS
    J647M2-17VH 857 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFLTTVIVTDNAMDYWGQG
    TTVTVSS
    J647M2-19VH 858 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFFTPVVVTDNAMDYWGQG
    TTVTVSS
    J647M2-1VH 859 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLMTTVVVTDHAMDYWGQG
    TTVTVSS
    J647M2-20VH 860 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKYLTTVVVTDSAMDYWGQG
    TTVTVSS
    J647M2-21VH 861 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFRSSVAVTDNAMDYWGQG
    TTVTVSS
    J647M2-22VH 862 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLFTTVVVTDSAMDYWGQG
    TTVTVSS
    J647M2-23VH 863 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKYLMPVVVTDYAMDYWGQG
    TTVTVSS
    J647M2-24VH 864 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKLLDAVMVTDYAMDYWGQG
    TTVTVSS
    J647M2-26VH 865 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFLTTVVVNDYAMDYWGQG
    TTVTVSS
    J647M2-44VH 866 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLLTTVAVTDYAMDYWGQG
    TTVTVSS
    J647M2-45VH 867 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFLKTVVATDDAMDYWGQG
    TTVTVSS
    J647M2-47VH 868 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFLNTAVVTDYAMDYWGQG
    TTVTVSS
    J647M2-48VH 869 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARRFLTTVDVTDNAMDYWGQG
    TTVTVSS
    J647M2-4VH 870 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKYLTPVVATDFAMDYWGQG
    TTVTVSS
    J647M2-51VH 871 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKCMTTIVETDNAMDYWGQG
    TTVTVSS
    J647M2-52VH 872 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFMNTVDVTDNAMDYWGQG
    TTVTVSS
    J647M2-53VH 873 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLFTTVVVTDDAMDYWGQG
    TTVTVSS
    J647M2-54VH 874 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLMTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2-55VH 875 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFLPTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2-56VH 876 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKLLTTVVVTDNAMDYWGQG
    TTVTVSS
    J647M2-58VH 877 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKILTTVVVTDNAMDYWGQG
    TTVTVSS
    J647M2-70VH 878 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKVMATEVVTDYAMDYWGQG
    TTVTVSS
    J647M2-71VH 879 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLVTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2-72VH 880 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFRKPVSVTDYAMDYWGQG
    TTVTVSS
    J647M2-73VH 881 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLWTTVVVTDNAMDYWGQG
    TTVTVSS
    J647M2-74VH 882 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLLTPVVVTDYAMDYWGQG
    TTVTVSS
    J647M2-75VH 883 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFRTTVVETDYCMDYWGQG
    TTVTVSS
    J647M2-76VH 884 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKYFTTVAVTDYAMDYWGQG
    TTVTVSS
    J647M2-78VH 885 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARRFLTTVEVTDLAMDYWGQG
    TTVTVSS
    J647M2-79VH 886 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFLRTEVMTDYAMDYWGQG
    TTVTVSS
    J647M2-7VH 887 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFLSTVAVTDSAMDYWGQG
    TTVTVSS
    J647M2-80VH 888 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKVLNTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2-83VH 889 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFMNTAMVTDYAMDYWGQG
    TTVTVSS
    J647M2-84VH 890 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFSTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2-85VH 891 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKYFTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2-86VH 892 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFLNTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-12VH 893 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFMPTVVETDYAMDYWGQG
    TTVTVSS
    J647M2S1-13VH 894 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGNPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-14VH 895 EVQLVQSGAEVKKPGASVKVSCKASGYTFADYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-15VH 896 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFLTTVVVTDCAMDYWGQG
    TTVTVSS
    J647M2S1-17VH 897 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-18VH 898 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFLTTVVVTDNAMDYWGQG
    TTVTVSS
    J647M2S1-19VH 899 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLLNTVVGTDYAMDYWGQG
    TTVTVSS
    J647M2S1-21VH 900 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKLLTTEAVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-22VH 901 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKYSTPVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-23VH 902 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGEPTYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-26VH 903 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKCLNTVAVTEHRMDYWGQG
    TTVTVSS
    J647M2S1-28VH 904 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFLTTVVHTDYAMDYWGQG
    TTVTVSS
    J647M2S1-30VH 905 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGQPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-31VH 906 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-32VH 907 EVQLVQSGAEVKKPGASVKVSCKASGYTFANYGINWVRQ
    APGQGLEWMGWINTYTGEPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-33VH 908 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFRTTVVLTDSAMDYWGQG
    TTVTVSS
    J647M2S1-35VH 909 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGINWVRQ
    APGQGLEWMGWINTYTGEPTYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-36VH 910 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFQTPVVDTDYAMDYWGQG
    TTVTVSS
    J647M2S1-39VH 911 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFMKTRVVTDNAMDYWGQG
    TTVTVSS
    J647M2S1-40VH 912 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGIVWVRQ
    APGQGLEWMGWINTYTGEPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-41VH 913 EVQLVQSGAEVKKPGASVKVSCKASGYTFPNYGISWVRQ
    APGQGLEWMGWINTYTGEPSYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-43VH 914 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGEPSYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-45VH 915 EVQLVQSGAEVKKPGASVKVSCKASGYTFTKYGINWVRQ
    APGQGLEWMGWINTYTGEPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-47VH 916 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKYLTTVVATDYAMDYWGQG
    TTVTVSS
    J647M2S1-48VH 917 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLLNTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-65VH 918 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFLTPVVVTDCAMDYWGQG
    TTVTVSS
    J647M2S1-66VH 919 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGEPRYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-67VH 920 EVQLVQSGAEVKKPGASVKVSCKASGYTFRDYGINWVRQ
    APGQGLEWMGWINTYTGLPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-69VH 921 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFWTTIVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-6VH 922 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKLLTTVSATDNAMDYWGQG
    TTVTVSS
    J647M2S1-70VH 923 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFLNTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-72VH 924 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGINWVRQ
    APGQGLEWMGWINTYNGEPSYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-75VH 925 EVQLVQSGAEVKKPGASVKVSCKASGYTFATYGIAWVRQ
    APGQGLEWMGWINTYSGVPKYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-76VH 926 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFRTTAVPTDNAMDYWGQG
    TTVTVSS
    J647M2S1-77VH 927 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFLTTVVNTDSAMDYWGQG
    TTVTVSS
    J647M2S1-78VH 928 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGRG
    TTVTVSS
    J647M2S1-79VH 929 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLLKTRVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-7VH 930 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-80VH 931 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLLTTVVATDYAMDYWGQG
    TTVTVSS
    J647M2S1-84VH 932 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGEPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-85VH 933 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGQPTYAQGFTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-87VH 934 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFFPTMVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-88VH 935 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKFVTTMVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-8VH 936 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYAQGLTGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFLTTVVVTDYAMDYWGQG
    TTVTVSS
    J647M2S1-92VH 937 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYFCARKLLTTIVATDNAMDYWGQG
    TTVTVSS
    J647M2S1-93VH 938 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLMSTVVETDNAMDYWGQG
    TTVTVSS
    J647M2S1-94VH 939 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLLFTVVQTDYAMDYWGQG
    TTVTVSS
    J647M2S1-96VH 940 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQ
    APGQGLEWMGWINTYTGEPTYADDFKGRFTFTLDTSTST
    AYMELSSLRSEDTAVYYCARKLLNTVVDTDYAMDYWGQG
    TTVTVSS
    J662M2S3-14VH 941 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGIIWVRQ
    APGQGLEWMGWINTYTGEPHYAQKLQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKLFTVEDVTDCAMDYWGQG
    TTVTVSS
    J662M2S3-18VH 942 EVQLVQSGAEVKKPGASVKVSCKASGYTFDNYGMNWVRQ
    APGQGLEWMGWINTYNGKPTYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKLFLVEAVTDYAMDYWGQG
    TTVTVSS
    J662M2S3-28VH 943 EVQLVQSGAEVKKPGASVKVSCKASGYTFRNYGIIWVRQ
    APGQGLEWMGWINTYTGKPTYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKLFTTVDVTDNAMDYWGQG
    TTVTVSS
    J662M2S3-29VH 944 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGIIWVRQ
    APGQGLEWMGWINTYTGVPTYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYYCARKLFNTVDVTDNAMDYWGQG
    TTVTVSS
    J662M2S3-30VH 945 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGIIWVRQ
    APGQGLEWMGWINTYTGEPHYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYYCARKLFKTMAVTDAAMDYWGQG
    TTVTVSS
    J662M2S3-34VH 946 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFRNTVAVTDYAMDYWGQG
    TTVTVSS
    J662M2S3-3VH 947 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGIIWVRQ
    APGQGLEWMGWINTYTGKPTYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYYCARKLFNTVAVTDNAMDYWGQG
    TTVTVSS
    J662M2S3-41VH 948 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYYCARKLFFTEDVTDYAMDYWGQG
    TTVTVSS
    J662M2S3-45VH 949 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGINWVRQ
    APGQGLEWMGWINTYTGKPTYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKFFTPVVVTDNAMDYWGQG
    TTVTVSS
    J662M2S3-55VH 950 EVQLVQSGAEVKKPGASVKVSCKASGYTFRNYGITWVRQ
    APGQGLEWMGWINTYTGKPTYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKLFTTMDVTDNAMDYWGQG
    TTVTVSS
    J662M2S3-5VH 951 EVQLVQSGAEVKKPGASVKVSCKASGYTFANYGIIWVRQ
    APGQGLEWMGWINTYTGKPTYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYYCARKLFTTMDVTDNAMDYWGQG
    TTVTVSS
    J662M2S3-65VH 952 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGIIWVRQ
    APGQGLEWMGWINTYTGKPTYAQKLQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKLFNTVDVTDNAMDYWGQG
    TTVTVSS
    J662M2S3-78VH 953 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGIIWVRQ
    APGQGLEWMGWINTYTGKPSYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYYCARKLFNTVDVTDNAMDYWGQG
    TTVTVSS
    J662M2S3-84VH 954 EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGINWVRQ
    APGQGLEWMGWINTYTGQPSYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYYCARKLFKTEAVTDYAMDYWGQG
    TTVTVSS
    J662M2S3-87VH 955 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGIIWVRQ
    APGQGLEWMGWINTYSGKPTYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYFCARKLFTTMDVTDNAMDYWGQG
    TTVTVSS
    J662M2S3-96VH 956 EVQLVQSGAEVKKPGASVKVSCKASGYTFNNYGIIWVRQ
    APGQGLEWMGWINTYTGKPTYAQKFQGRVTMTTDTSTST
    AYMELSSLRSEDTAVYYCARKFFTTMAVTDNAMDYWGQG
    TTVTVSS
  • Table 28 provides a list of amino acid sequences of VL regions of affinity matured fully human TNF antibodies derived from hMAK199 Amino acid residues of individual CDRs of each VL sequence are indicated in bold.
  • TABLE 28
    List of amino acid sequences
    of affinity matured hMAK199 VL variants
    Clone SEQ ID NO: VL
    J644M2S1-11Vk 957 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTLPPTFGQGTKLEIK
    J644M2S1-73Vk 958 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J647M2-11Vk 959 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKTVKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J647M2S1-10Vk 960 DIQMTQSPSSLSASVGDRVTITCRASQDIWNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNRYPPTFGQGTKLEIK
    J647M2S1-16Vk 961 DIQMTQSPSSLSASVGDRVTITCRASQDICTYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNSPPPTFGQGTKLEIK
    J647M2S1-1Vk 962 DIQMTQSPSSLSASVGDRVTITCRASQAIGNYLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J647M2S1-20Vk 963 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTRPPTFGQGTKLEIK
    J647M2S1-24Vk 964 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSLLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTGPPTFGQGTKLEIK
    J647M2S1-25Vk 965 DIQMTQSPSSLSASVGDRVTITCRASQDIYNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J647M2S1-29Vk 966 DIQMTQSPSSLSASVGDRVTITCRASQDISHYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTLPATFGQGTKLEIK
    J647M2S1-2Vk 967 DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQK
    PGKTVKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTPPPTFGQGTKLEIK
    J647M2S1-34Vk 968 DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTLPPTFGQGTKLEIK
    J647M2S1-37Vk 969 DIQMTQSPSSLSASVGDRVTITCRASQEISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTMPTTFGQGTKLEIK
    J647M2S1-38Vk 970 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYFASRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTPPTTFGQGTKLEIK
    J647M2S1-3Vk 971 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTLPSTFGQGTKLEIK
    J647M2S1-42Vk 972 DIQMTQSPSSLSASVGDRVTITCRASQVISNTLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNALPPTFGQGTKLEIK
    J647M2S1-44Vk 973 DIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTPPPTFGQGTKLEIK
    J647M2S1-46Vk 974 DIQMTQSPSSLSASVGDRVTITCRASQDISQYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTLPPTFGQGTKLEIK
    J647M2S1-50Vk 975 DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTAPPTFGQGTKLEIK
    J647M2S1-52Vk 976 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTMPPTFGQGTKLEIK
    J647M2S1-56Vk 977 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J647M2S1-59Vk 978 DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTRPPTFGQGTKLEIK
    J647M2S1-71Vk 979 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSLLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTQPPTFGQGTKLEIK
    J647M2S1-74Vk 980 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNSQPPTFGQGTKLEIK
    J647M2S1-78Vk 981 DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQK
    PGKAPKLLIYNASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J647M2S1-7Vk 982 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSLLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNIWPPTFGQGTKLEIK
    J647M2S1-9Vk 983 DIQMTQSPSSLSASVGDRVTITCRASQDISHYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J652M2S1-10Vk 984 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTFPPTFGQGTKLEIK
    J652M2S1-13Vk 985 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTQPPTFGQGTKLEIK
    J652M2S1-14Vk 986 DIQMTQSPSSLSASVGDRVTITCRASQDISNVLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTLPPTFGQGTKLEIK
    J652M2S1-15Vk 987 DIQMTQSPSSLSASVGDRVTITCRASQDIYKYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTMPPTFGQGTKLEIK
    J652M2S1-17Vk 988 DIQMTQSPSSLSASVGDRVTITCRASQEIFSYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNMGPPTFGQGTKLEIK
    J652M2S1-18Vk 989 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTQPPTFGQGTKLEIK
    J652M2S1-1Vk 990 DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTWPPTFGQGTKLEIK
    J652M2S1-22Vk 991 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTRPPTFGQGTKLEIK
    J652M2S1-23Vk 992 DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTFPPTFGQGTKLEIK
    J652M2S1-25Vk 993 DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTRPPTFGQGTKLEIK
    J652M2S1-26Vk 994 DIQMTQSPSSLSASVGDRVTITCRASQDINNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTQPPTFGQGTKLEIK
    J652M2S1-27Vk 995 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYASGLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTWPPTFGQGTKLEIK
    J652M2S1-28Vk 996 DIQMTQSPSSLSASVGDRVTITCRASQDISRYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTQPPTFGQGTKLEIK
    J652M2S1-29Vk 997 DIQMTQSPSSLSASVGDRVTITCRASQDIATYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTMPPTFGQGTKLEIK
    J652M2S1-31Vk 998 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTFPPTFGQGTKLEIK
    J652M2S1-33Vk 999 DIQMTQSPSSLSASVGDRVTITCRASQRIGNYLNWYQQK
    PGKTVKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J652M2S1-34Vk 1000 DIQMTQSPSSLSASVGDRVTITCRASQEISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNSQPPTFGQGTKLEIK
    J652M2S1-35Vk 1001 DIQMTQSPSSLSASVGDRVTITCRASQDIANYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTLPPTFGQGTKLEIK
    J652M2S1-37Vk 1002 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTFPPTFGQGTKLEIK
    J652M2S1-38Vk 1003 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTQPPTFGQGTKLEIK
    J652M2S1-3Vk 1004 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTPPPTFGQGTKLEIK
    J652M2S1-40Vk 1005 DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTLPPTFGQGTKLEIK
    J652M2S1-41Vk 1006 DIQMTQSPSSLSASVGDRVTITCRASQDIGNFLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTRPPTFGQGTKLEIK
    J652M2S1-42Vk 1007 DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTPPPTFGQGTKLEIK
    J652M2S1-45Vk 1008 DIQMTQSPSSLSASVGDRVTITCRASQDISDYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNMWPPTFGQGTKLEIK
    J652M2S1-47Vk 1009 DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTMPPTFGQGTKLEIK
    J652M2S1-48Vk 1010 DIQMTQSPSSLSASVGDRVTITCRASQDISHYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTLPPTFGQGTKLEIK
    J652M2S1-49Vk 1011 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTMPPTFGQGTKLEIK
    J652M2S1-51Vk 1012 DIQMTQSPSSLSASVGDRVTITCRASQDISQYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTRPPTFGQGTKLEIK
    J652M2S1-52Vk 1013 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNMRPPTFGQGTKLEIK
    J652M2S1-53Vk 1014 DIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J652M2S1-55Vk 1015 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTGPPTFGQGTKLEIK
    J652M2S1-56Vk 1016 DIQMTQSPSSLSASVGDRVTITCRASQNINNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTMPPTFGQGTKLEIK
    J652M2S1-57Vk 1017 DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTPPPTFGQGTKLEIK
    J652M2S1-61Vk 1018 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTVPPTFGQGTKLEIK
    J652M2S1-62Vk 1019 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSKLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNIFPPTFGQGTKLEIK
    J652M2S1-64Vk 1020 DIQMTQSPSSLSASVGDRVTITCRASQGIYNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J652M2S1-67Vk 1021 DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTLPPTFGQGTKLEIK
    J652M2S1-69Vk 1022 DIQMTQSPSSLSASVGDRVTITCRASQEISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTGPPTFGQGTKLEIK
    J652M2S1-6Vk 1023 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTPPPTFGQGTKLEIK
    J652M2S1-71Vk 1024 DIQMTQSPSSLSASVGDRVTITCRASQDISDYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTWPPTFGQGTKLEIK
    J652M2S1-73Vk 1025 DIQMTQSPSSLSASVGDRVTITCRASQDIWKYLNWYQQK
    PGKAPKLLIYYASRLQSVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTLPPTFGQGTKLEIK
    J652M2S1-75Vk 1026 DIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTWPPTFGQGTKLEIK
    J652M2S1-77Vk 1027 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTPPPTFGQGTKLEIK
    J652M2S1-78Vk 1028 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNAPPPTFGQGTKLEIK
    J652M2S1-79Vk 1029 DIQMTQSPSSLSASVGDRVTITCRASQDIYKFLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J652M2S1-80Vk 1030 DIQMTQSPSSLSASVGDRVTITCRASQDIFNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J652M2S1-82Vk 1031 DIQMTQSPSSLSASVGDRVTITCRASQDISNTLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTLPPTFGQGTKLEIK
    J652M2S1-84Vk 1032 DIQMTQSPSSLSASVGDRVTITCRASQHISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTQPPTFGQGTKLEIK
    J652M2S1-86Vk 1033 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNMPPPTFGQGTKLEIK
    J652M2S1-87Vk 1034 DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTVPPTFGQGTKLEIK
    J652M2S1-8Vk 1035 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYFTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGNTQPPTFGQGTKLEIK
    J652M2S1-90Vk 1036 DIQMTQSPSSLSASVGDRVTITCRASQDISKFLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYYCQQGNTRPPTFGQGTKLEIK
    J652M2S1-91Vk 1037 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTFPPTFGQGTKLEIK
    J652M2S1-92Vk 1038 DIQMTQSPSSLSASVGDRVTITCRASQDIYNVLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGITLPPTFGQGTKLEIK
    J652M2S1-93Vk 1039 DIQMTQSPSSLSASVGDRVTITCRASQHISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTWPPTFGQGTKLEIK
    J652M2S1-95Vk 1040 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTQPSTFGQGTKLEIK
    J652M2S1-9Vk 1041 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTQPPTFGQGTKLEIK
    J662M2S3-13Vk 1042 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNSWPPTFGQGTKLEIK
    J662M2S3-15Vk 1043 DIQMTQSPSSLSASVGDRVTITCRASQDIYNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTQPPTFGQGTKLEIK
    J662M2S3-21Vk 1044 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTWPPTFGQGTKLEIK
    J662M2S3-22Vk 1045 DIQMTQSPSSLSASVGDRVTITCRASQDISQYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTWPPTFGQGTKLEIK
    J662M2S3-34Vk 1046 DIQMTQSPSSLSASVGDRVTITCRASQDIYDVLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYYCQQGITLPPTFGQGTKLEIK
    J662M2S3-3Vk 1047 DIQMTQSPSSLSASVGDRVTITCRASQDIENYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTQPPTFGQGTKLEIK
    J662M2S3-41Vk 1048 DIQMTQSPSSLSASVGDRVTITCRASQNIENFLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTWPPTFGQGTKLEIK
    J662M2S3-56Vk 1049 DIQMTQSPSSLSASVGDRVTITCRASQDIYNYLNWYQQK
    PGKAPKLLIYYTSRLQGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTPPPTFGQGTKLEIK
    J662M2S3-64Vk 1050 DIQMTQSPSSLSASVGDRVTITCRASQDIASYLNWYQQK
    PGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTQPPTFGQGTKLEIK
    J662M2S3-78Vk 1051 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
    PGKVPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSL
    QPEDFATYFCQQGNTQPPTFGQGTKLEIK
    J662M2S3-84Vk 1052 DIQMTQSPSSLSASVGDRVTITCRASQNIYNVLNWYQQK
    PGKAPKLLIYYASRLQSGVPSRFSGSGSGTDFTLTISSL
    QPEDFATYFCQQGNTMPPTFGQGTKLEIK
  • TABLE 29
    Amino acid residues observed in affinity matured hMAK-199 antibodies
    MAK199 Heavy chain variable region (SEQ ID NO: 1077)
    MAK199VH.2a 1234567890123456789012345678901234567890123456789012a345678901
    EIQLVQSGAEVKKPGASVKVSCKASGYTFT NYGMN WVRQAPGQGLEWMG WINTYTGEPTYAD
     V                           ND  II                   N K S  Q
                                 AH   T                   S V H
                                 ST   Q                     Q N
                                 RS   S                     R M
                                 DQ   G                     L K
                                 KK   A                     S A
                                 P    V                     N R
                                 Q                          I Q
                                 M                          D D
                                 G                          A
                                 E
    234567890123456789012abc345678901234567890abcdefg1234567890123
    DFKG RFTFTLDTSTSTAYMELSSLRSEDTAVYFCAR KFLTTVVVTDYAMDY WGQGTTVTVSS
    GLT  V M T                      Y   RLFNPMDASENT
    K Q                                 NYMKVEAEM SR
                                         IRSSAEMN CC
                                         VSRARSD  H
                                         CWL IMG  D
                                          QP QII  I
                                          VF GPQ  F
                                          ND D P  V
                                          GM   N  L
                                          CA   L  A
                                               H
    Mak199 Light chain variable region (SEQ ID NO: 1078)
    Mak199Vk.1a 1234567890123456789012345678901234567890123456789012345678901
    DIQMTQSPSSLSASVGDRVTITC RASQDISNYLN WYQQKPGKTVKLLIY YTSRLQS GVPSR
                               N YQV          AP     FA L
                               E ESF          V      N  K
                               H AKT                    G
                               G TT
                               V WH
                               R GD
                               A NR
                                 F
                                 C
    234567890123456789012345678901234567890123456a
    FSGSGSGTDYTLTISSLQPEDFATYFC QQGNTLPPT FGQGTKLEIK
             F               Y    ISW T
                                   MQ S
                                   IP A
                                   AM
                                   RR
                                    F
                                    G
                                    V
                                    Y
                                    A
  • TABLE 30
    Individual hMAK-199 VH sequences from converted clones
    Protein Sequence
    region SEQ ID NO: 123456789012345678901234567890
    J662M2S3 1053 EVQLVQSGAEVKKPGASVKVSCKASGYTFA
    #10 VH NYGIIWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYYCARKLFTTMDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    10 VH of SEQ ID
    NO.: 1053
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    10 VH of SEQ ID
    NO.: 1053
    J662M2S3# CDR-H3 Residues 99-112 RASQDISQYLN
    10 VH of SEQ ID
    NO.: 1053
    J662M2S3# 1054 EVQLVQSGAEVKKPGASVKVSCKASGYTFN
    13 VH NYGIIWVRQAPGQGLEWMGWINTYTGKPTY
    AQKLQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYFCARKLFNTVDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    13 VH of SEQ ID
    NO.: 1054
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKLQG
    13 VH of SEQ ID
    NO.: 1054
    J662M2S3# CDR-H3 Residues 99-112 KLFNTVDVTDNAMD
    13 VH of SEQ ID
    NO.: 1054
    J662M2S3# 1055 EVQLVQSGAEVKKPGASVKVSCKASGYTFN
    15 VH NYGIIWVRQAPGQGLEWMGWINTYTGVPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYYCARKLFNTVDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    15 VH of SEQ ID
    NO.: 1055
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGVPTYAQKFQG
    15 VH of SEQ ID
    NO.: 1055
    J662M2S3# CDR-H3 Residues 99-112 KLFNTVDVTDNAMD
    15 VH of SEQ ID
    NO.: 1055
    J662M2S3# 1056 EVQLVQSGAEVKKPGASVKVSCKASGYTFN
    16 VH NYGIIWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYYCARKLFNTVAVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    16 VH of SEQ ID
    NO.: 1056
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    16 VH of SEQ ID
    NO.: 1056
    J662M2S3# CDR-H3 Residues 99-112 KLFNTVAVTDNAMD
    16 VH of SEQ ID
    NO.: 1056
    J662M2S3# 1057 EVQLVQSGAEVKKPGASVKVSCKASGYTFR
    21 VH NYGIIWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYFCARKLFTTVDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    21 VH of SEQ ID
    NO.: 1057
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    21 VH of SEQ ID
    NO.: 1057
    J662M2S3# CDR-H3 Residues 99-112 KLFTTVDVTDNAMD
    21 VH of SEQ ID
    NO.: 1057
    J662M2S3# 1058 EVQLVQSGAEVKKPGASVKVSCKASGYTFN
    34 VH NYGINWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYFCARKFRNTVAVTDYAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGIN
    34 VH of SEQ ID
    NO.: 1058
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    34 VH of SEQ ID
    NO.: 1058
    J662M2S3# CDR-H3 Residues 99-112 KFRNTVAVTDYAMD
    34 VH of SEQ ID
    NO.: 1058
    J662M2S3# 1059 EVQLVQSGAEVKKPGASVKVSCKASGYTFR
    36 VH NYGITWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYFCARKLFTTMDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGIT
    36 VH of SEQ ID
    NO.: 1059
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    36 VH of SEQ ID
    NO.: 1059
    J662M2S3# CDR-H3 Residues 99-112 KLFTTMDVTDNAMD
    36 VH of SEQ ID
    NO.: 1059
    J662M2S3# 1060 EVQLVQSGAEVKKPGASVKVSCKASGYTFA
    45 VH NYGIIWVRQAPGQGLEWMGWINTYTGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYYCARKLFTTMDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    45 VH of SEQ ID
    NO.: 1060
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGKPTYAQKFQG
    45 VH of SEQ ID
    NO.: 1060
    J662M2S3# CDR-H3 Residues 99-112 KLFTTMDVTDNAMD
    45 VH of SEQ ID
    NO.: 1060
    J662M2S3# 1061 EVQLVQSGAEVKKPGASVKVSCKASGYTFS
    58 VH NYGINWVRQAPGQGLEWMGWINTYTGQPSY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYYCARKLFKTEAVTDYAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGIN
    58 VH of SEQ ID
    NO.: 1061
    J662M2S3# CDR-H2 Residues 50-66 WINTYTGQPSYAQKFQG
    58 VH of SEQ ID
    NO.: 1061
    J662M2S3# CDR-H3 Residues 99-112 KLFKTEAVTDYAMD
    58 VH of SEQ ID
    NO.: 1061
    J662M2S3# 1062 EVQLVQSGAEVKKPGASVKVSCKASGYTFN
    72 VH NYGIIWVRQAPGQGLEWMGWINTYSGKPTY
    AQKFQGRVTMTTDTSTSTAYMELSSLRSED
    TAVYFCARKLFTTMDVTDNAMDYWGQGTTV
    TVSS
    J662M2S3# CDR-H1 Residues 31-35 NYGII
    72 VH of SEQ ID
    NO.: 1062
    J662M2S3# CDR-H2 Residues 50-66 WINTYSGKPTYAQKFQG
    72 VH of SEQ ID
    NO.: 1062
    J662M2S3# CDR-H3 Residues 99-112 KLFTTMDVTDNAMD
    72 VH of SEQ ID
    NO.: 1062
  • TABLE 31
    Individual hMAK-199 clones VL sequences
    Protein Sequence
    region SEQ ID NO: 123456789012345678901234567890
    J662M2S3# 1063 DIQMTQSPSSLSASVGDRVTITCRASQDIS
    10 VL QYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
    GNTWPPTFGQGTKLEIK
    J662M2S3#10 CDR-L1 Residues 24-34 RASQDISQYLN
    VL of SEQ ID
    NO.: 1063
    J662M2S3#10 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 1063
    J662M2S3#10 CDR-L3 Residues 89-97 QQGNTWPPT
    VL of SEQ
    ID NO.: 1063
    J662M2S3#13 1064 DIQMTQSPSSLSASVGDRVTITCRASQDIS
    VL NYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDYTLTISSLQPEDFATYFCQQ
    GNSWPPTFGQGTKLEIK
    J662M2S3#13 CDR-L1 Residues 24-34 RASQDISNYLN
    VL of SEQ ID
    NO.: 1064
    J662M2S3#13 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 1064
    J662M2S3#13 CDR-L3 Residues 89-97 QQGNSWPPT
    VL of SEQ
    ID NO.: 1064
    J662M2S3#15 1065 DIQMTQSPSSLSASVGDRVTITCRASQDIY
    VL NYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDYTLTISSLQPEDFATYFCQQ
    GNTQPPTFGQGTKLEIK
    J662M2S3#15 CDR-L1 Residues 24-34 RASQDIYNYLN
    VL of SEQ ID
    NO.: 1065
    J662M2S3#15 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 1065
    J662M2S3#15 CDR-L3 Residues 89-97 QQGNTQPPT
    VL of SEQ
    ID NO.: 1065
    J662M2S3#16 1066 DIQMTQSPSSLSASVGDRVTITCRASQDIE
    VL NYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
    GNTQPPTFGQGTKLEIK
    J662M2S3#16 CDR-L1 Residues 24-34 RASQDIENYLN
    VL of SEQ ID
    NO.: 1066
    J662M2S3#16 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 1066
    J662M2S3#16 CDR-L3 Residues 89-97 QQGNTQPPT
    VL of SEQ
    ID NO.: 1066
    J662M2S3#21 1067 DIQMTQSPSSLSASVGDRVTITCRASQDIS
    VL NYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDYTLTISSLQPEDFATYFCQQ
    GNTWPPTFGQGTKLEIK
    J662M2S3#21 CDR-L1 Residues 24-34 RASQDISNYLN
    VL of SEQ ID
    NO.: 1067
    J662M2S3#21 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 1067
    J662M2S3#21 CDR-L3 Residues 89-97 QQGNTWPPT
    VL of SEQ
    ID NO.: 1067
    J662M2S3#34 1068 DIQMTQSPSSLSASVGDRVTITCRASQDIY
    VL DVLNWYQQKPGKAPKLLIYYASRLQSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
    GITLPPTFGQGTKLEIK
    J662M2S3#34 CDR-L1 Residues 24-34 RASQDIYDVLN
    VL of SEQ ID
    NO.: 1068
    J662M2S3#34 CDR-L2 Residues 50-56 YASRLQS
    4 VL of SEQ ID
    NO.: 1068
    J662M2S3#34 CDR-L3 Residues 89-97 QQGITLPPT
    VL of SEQ
    ID NO.: 1068
    J662M2S3#36 1069 DIQMTQSPSSLSASVGDRVTITSRASQDIS
    VL NYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDYTLTISSLQPEDFATYFCQQ
    GNTWPPTFGQGTKLEIK
    J662M2S3#36 CDR-L1 Residues 24-34 RASQDISNYLN
    VL of SEQ ID
    NO.: 1069
    J662M2S3#36 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 1069
    J662M2S3#36 CDR-L3 Residues 89-97 QQGNTWPPT
    VL of SEQ
    ID NO.: 1069
    J662M2S3#45 1070 DIQMTQSPSSLSASVGDRVTITSRASQDIS
    VL QYLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
    GNTWPPTFGQGTKLEIK
    J662M2S3#45 CDR-L1 Residues 24-34 RASQDISQYLN
    VL of SEQ ID
    NO.: 1070
    J662M2S3#45 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 1070
    J662M2S3#45 CDR-L3 Residues 89-97 QQGNTWPPT
    VL of SEQ
    ID NO.: 1070
    J662M2S3#58 1071 DIQMTQSPSSLSASVGDRVTITSRASQNIY
    VL NVLNWYQQKPGKAPKLLIYYASRLQSGVPS
    RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
    GNTMPPTFGQGTKLEIK
    J662M2S3#58 CDR-L1 Residues 24-34 RASQNIYNVLN
    VL of SEQ ID
    NO.: 1071
    J662M2S3#58 CDR-L2 Residues 50-56 YASRLQS
    VL of SEQ ID
    NO.: 1071
    J662M2S3#58 CDR-L3 Residues 89-97 QQGNTMPPT
    VL of SEQ
    ID NO.: 1071
    J662M2S3#72 1072 DIQMTQSPSSLSASVGDRVTITSRASQDIS
    VL NFLNWYQQKPGKAPKLLIYYTSRLQSGVPS
    RFSGSGSGTDYTLTISSLQPEDFATYFCQQ
    GNTQPPTFGQGTKLEIK
    J662M2S3#72 CDR-L1 Residues 24-34 RASQDISNFLN
    VL of SEQ ID
    NO.: 1072
    J662M2S3#72 CDR-L2 Residues 50-56 YTSRLQS
    VL of SEQ ID
    NO.: 1072
    J662M2S3#72 CDR-L3 Residues 89-97 QQGNTQPPT
    VL of SEQ
    ID NO.: 1072
  • TABLE 32
    hMAK199 affinity matured scFv clones converted to full length IgG
    ScFv Full length IgG
    clone name HC plasmid LC plasmid (protein) name
    J662M2S3#10 pHybE-hCg1,z,non-a V2 pHybE-hCk V3 J662 hMAK199-AM1
    J662M2S3#10 M2S3#10
    J662M2S3#13 pHybE-hCg1,z,non-a V2 pHybE-hCk V3 J662 hMAK199-AM2
    J662M2S3#13 M2S3#13
    J662M2S3#15 pHybE-hCg1,z,non-a V2 pHybE-hCk V3 J662 hMAK199-AM3
    J662M2S3#15 M2S3#15
    J662M2S3#16 pHybE-hCg1,z,non-a V2 pHybE-hCk V3 J662 hMAK199-AM4
    J662M2S3#16 M2S3#16
    J662M2S3#21 pHybE-hCg1,z,non-a V2 pHybE-hCk V3 J662 hMAK199-AM5
    J662M2S3#21 M2S3#21
    J662M2S3#34 pHybE-hCg1,z,non-a V2 pHybE-hCk V3 J662 hMAK199-AM6
    J662M2S3#34 M2S3#34
    J662M2S3#36 pHybE-hCg1,z,non-a V2 pHybE-hCk V3 J662 hMAK199-AM7
    J662M2S3#36 M2S3#36
    J662M2S3#45 pHybE-hCg1,z,non-a V2 pHybE-hCk V3 J662 hMAK199-AM8
    J662M2S3#45 M2S3#45
    J662M2S3#58 pHybE-hCg1,z,non-a V2 pHybE-hCk V3 J662 hMAK199-AM9
    J662M2S3#58 M2S3#58
    J662M2S3#72 pHybE-hCg1,z,non-a V2 pHybE-hCk V3 J662 hMAK199-AM10
    J662M2S3#72 M2S3#72
    • 3.1 TNF Enzyme-Linked Immunosorbent Assay Result
  • TABLE 33
    hMAK199 affinity matured full length IgG
    IgG Name EC50 in hTNFa ELISA (nM)
    hMAK199-AM1 0.016
    hMAK199-AM2 0.016
    hMAK199-AM3 0.019
    hMAK199-AM4 0.050
    hMAK199-AM5 0.078
    hMAK199-AM6 0.035
    hMAK199-AM7 0.100
    hMAK199-AM8 0.219
    hMAK199-AM9 0.032
    hMAK199-AM10 0.014
    • 3.2 TNF Neutralization Potency of TNF Antibodies by L929 Bioassay
  • TABLE 34
    hu TNF rhesus TNF neutralization
    IgG Name neutralization IC50 (nM) IC50 (nM)
    hMAK199-AM1 0.054 0.012
    hMAK199-AM2 0.029 0.010
    hMAK199-AM3 0.051 0.019
    hMAK199-AM4 0.028 0.005
    hMAK199-AM5 0.087 0.020
    hMAK199-AM6 0.033 0.004
    hMAK199-AM7 0.095 0.051
    hMAK199-AM8 0.247 0.204
    hMAK199-AM9 0.163 0.089
    hMAK199-AM10 0.048 0.034
    • Example 4 Example 4.4 Affinity Determination Using BIACORE Technology
  • TABLE 35
    Reagent for Biacore Analyses
    Antigen Vendor Designation Vendor Catalog #
    TNFα Recombinant Human TNF- R&D 210-TA
    α/TNFSF1A systems
    • BIACORE Methods:
  • The BIACORE assay (Biacore, Inc. Piscataway, N.J.) determines the affinity of binding proteins with kinetic measurements of on-rate and off-rate constants. Binding of binding proteins to a target antigen (for example, a purified recombinant target antigen) is determined by surface plasmon resonance-based measurements with a Biacore® 1000 or 3000 instrument (Biacore®AB, Uppsala, Sweden) using running HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25° C. All chemicals are obtained from Biacore® AB (Uppsala, Sweden) or otherwise from a different source as described in the text. For example, approximately 5000 RU of goat anti-mouse IgG, (Fey), fragment specific polyclonal antibody (Pierce Biotechnology Inc, Rockford, Ill., US) diluted in 10 mM sodium acetate (pH 4.5) is directly immobilized across a CM5 research grade biosensor chip using a standard amine coupling kit according to manufacturer's instructions and procedures at 25 μg/ml. Unreacted moieties on the biosensor surface are blocked with ethanolamine. Modified carboxymethyl dextran surface in flowcell 2 and 4 is used as a reaction surface. Unmodified carboxymethyl dextran without goat anti-mouse IgG in flow cell 1 and 3 is used as the reference surface. For kinetic analysis, rate equations derived from the 1:1 Langmuir binding model are fitted simultaneously to association and dissociation phases of all eight injections (using global fit analysis) with the use of Biaevaluation 4.0.1 software. Purified antibodies are diluted in HEPES-buffered saline for capture across goat anti-mouse IgG specific reaction surfaces. Antibodies to be captured as a ligand (25 μg/ml) are injected over reaction matrices at a flow rate of 5 μl/minute. The association and dissociation rate constants, kon (M1 s1) and koff (s−1), are determined under a continuous flow rate of 25 μl/minute. Rate constants are derived by making kinetic binding measurements at different antigen concentrations ranging from 10-200 nM. The equilibrium dissociation constant (M) of the reaction between antibodies and the target antigen is then calculated from the kinetic rate constants by the following formula: KD=koff/kon. Binding is recorded as a function of time and kinetic rate constants are calculated. In this assay, on-rates as fast as 106M−1 s−1 and off-rates as slow as 10−6 s−1 can be measured.
  • The binding proteins herein are expected to have beneficial properties in this regard, including high affinity, slow off rate, and high neutralizing capacity.
    • Example 4.5 Neutralization of Human TNF-α
  • L929 cells are grown to a semi-confluent density and harvested using 0.25% trypsin
  • (Gibco#25300). The cells are washed with PBS, counted and resuspended at 1E6 cells/mL in assay media containing 4 μg/mL actinomycin D. The cells are seeded in a 96-well plate (Costar#3599) at a volume of 100 μL and 5E4 cells/well. The binding proteins and control IgG are diluted to a 4× concentration in assay media and serial 1:4 dilutions are performed. The huTNF-α is diluted to 400 pg/mL in assay media. Binding protein sample (200 μL) is added to the huTNF-α (200 μL) in a 1:2 dilution scheme and allowed to incubate for 0.5 hour at room temperature.
  • The binding protein/human TNF-α solution is added to the plated cells at 100 μL for a final concentration of 100 pg/mL huTNF-α and 150 nM-0.0001 nM binding protein. The plates are incubated for 20 hours at 37° C., 5% CO2. To quantitate viability, 100 μL is removed from the wells and 10 μL of WST-1 reagent (Roche cat#11644807001) is added. Plates are incubated under assay conditions for 3.5 hours. The plates are read at OD 420-600 nm on a Spectromax 190 ELISA plate reader.
  • The binding proteins herein are expected to have beneficial properties in this regard, including high affinity, slow off rate, and high neutralizing capacity.
    • Example 4.6 Treatment
  • A patient requiring treatment with a TNF-α binding protein may have a disease with immune and inflammatory elements, such as autoimmune diseases, particularly those associated with inflammation, including Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, and ankylosing spondylitis. Therefore, the binding proteins herein may be used to treat these disorders.
  • Administration of the TNF-α binding protein may occur by subcutaneous injection. If the patient has rheumatoid arthritis, psoratic arthritis, or ankylosing spondyitis, the patient may receive 40 mg every other week as a starting dose and 40 mg every week, if necessary to achieve treatment goals. If the patient has juvenile idiopathic arthritis and weighs from 15 kg to <30 kg, the patient may receive 20 mg every other week, and if >30 kg, 40 mg every other week. If the patient has Crohn's disease, the patient may receive an initial dose of 160 mg (four 40 mg injections in one day or two 40 mg injections per day for two consecutive days) followed by 80 mg two weeks later, and another two weeks later begin a maintenance dose of 40 mg every other week. If the patient has plaque psoriasis, the patient may receive an 80 mg initial dose, followed by 40 mg every other week starting one week after initial dose.
  • The binding protein may be provided in a single-use prefilled pen (40 mg/0.8 mL), a single-use prefilled glass syringe (40 mg/0.8 mL or 20 mg/0.4 mL).
    • INCORPORATION BY REFERENCE
  • The contents of all cited references (including literature references, patents, patent applications, and websites) that are cited throughout this application are hereby expressly incorporated by reference in their entirety, as are the references cited therein. The practice disclosed herein will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology and cell biology, which are well known in the art.
    • EQUIVALENTS
  • The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.

Claims (36)

1-18. (canceled)
19. A method for treating a mammal comprising administering to the mammal an effective amount of a pharmaceutical composition comprising a binding protein that binds human TNFα, the binding protein comprising at least one heavy chain variable region (VH region) and at least one light chain variable region (VL region),
wherein the VH region comprises the amino acid sequences of the three complementarity determining regions (CDRs) from SEQ ID NO: 1077 or the amino acid sequence of SEQ ID NO: 1077,
wherein the VL region comprises the amino acid sequence of the three CDRs from SEQ ID NO: 1078 or the amino acid sequence of SEQ ID NO: 1078.
20. (canceled)
21. A method for reducing human TNF-α activity in a human subject suffering from a disorder in which TNF-α activity is detrimental, the method comprising administering to the human subject a binding protein of claim 19 such that human TNF-α activity in the human subject is reduced and/or treatment is achieved.
22. A method for treating a patient suffering from a disorder in which TNF-α is detrimental comprising administering to the patient the binding protein of claim 19 either before, concurrent, or after the administration to the patient of a second agent, wherein the second agent is selected from the group consisting of an antibody, or fragment thereof, capable of binding human IL-12; PGE2; LPA; NGF; CGRP; SubP; RAGE; histamine; a histamine receptor blocker; bradykinin; IL-1alpha; IL-1beta; VEGF; PLGF; methotrexate; a corticosteroid, a glucocorticoid receptor modulator; cyclosporin, rapamycin, FK506, and a non-steroidal anti-inflammatory agent.
23. The method of claim 21, wherein the disorder is an autoimmune and/or inflammatory disorder.
24. The method of claim 23, wherein the disorder is selected from the group consisting of Crohn's disease, psoriasis, plaque psoriasis, arthritis, rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis, multiple sclerosis, and ankylo sing spondylitis.
25. The method of claim 23, wherein the disorder is selected from the group consisting of a respiratory disorder; asthma; allergic and nonallergic asthma; asthma due to infection; asthma due to infection with respiratory syncytial virus (RSV); chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation; eosinophilia; fibrosis and excess mucus production; cystic fibrosis; pulmonary fibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema; allergic rhinitis; allergic enterogastritis; an inflammatory and/or autoimmune condition of the skin; an inflammatory and/or autoimmune condition of gastrointestinal organs; inflammatory bowel diseases (IBD); ulcerative colitis; Crohn's disease; an inflammatory and/or autoimmune condition of the liver; liver cirrhosis; liver fibrosis; liver fibrosis caused by hepatitis B and/or C virus; scleroderma; tumors or cancers; hepatocellular carcinoma; glioblastoma; lymphoma; Hodgkin's lymphoma; a viral infection; a bacterial infection; a parasitic infection; HTLV-1 infection; suppression of expression of protective type 1 immune responses, and suppression of expression of a protective type 1 immune response during vaccination.
26-29. (canceled)
30. The method of claim 22, wherein the disorder is an autoimmune and/or inflammatory disorder.
31. The method of claim 30, wherein the disorder is selected from the group consisting of Crohn's disease, psoriasis, plaque psoriasis, arthritis, rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis, multiple sclerosis, and ankylo sing spondylitis.
32. The method of claim 30, wherein the disorder is selected from the group consisting of a respiratory disorder; asthma; allergic and nonallergic asthma; asthma due to infection; asthma due to infection with respiratory syncytial virus (RSV); chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation; eosinophilia; fibrosis and excess mucus production; cystic fibrosis; pulmonary fibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema; allergic rhinitis; allergic enterogastritis; an inflammatory and/or autoimmune condition of the skin; an inflammatory and/or autoimmune condition of gastrointestinal organs; inflammatory bowel diseases (IBD); ulcerative colitis; Crohn's disease; an inflammatory and/or autoimmune condition of the liver; liver cirrhosis; liver fibrosis; liver fibrosis caused by hepatitis B and/or C virus; scleroderma; tumors or cancers; hepatocellular carcinoma; glioblastoma; lymphoma; Hodgkin's lymphoma; a viral infection; a bacterial infection; a parasitic infection; HTLV-1 infection; suppression of expression of protective type 1 immune responses, and suppression of expression of a protective type 1 immune response during vaccination.
33. A method for treating a mammal comprising administering to the mammal an effective amount of a pharmaceutical composition comprising a binding protein that binds human TNFα, the binding protein comprising at least one heavy chain variable region (VH region), wherein the VH region comprises the amino acid sequences of the three complementarity determining regions (CDRs) from SEQ ID NO: 1077 or the amino acid sequence of SEQ ID NO: 1077.
34. A method for reducing human TNF-α activity in a human subject suffering from a disorder in which TNF-α activity is detrimental, the method comprising administering to the human subject the binding protein of claim 33 such that human TNF-α activity in the human subject is reduced and/or treatment is achieved.
35. A method for treating a patient suffering from a disorder in which TNF-α is detrimental comprising administering to the patient the binding protein of claim 33 either before, concurrent, or after the administration to the patient of a second agent, wherein the second agent is selected from the group consisting of an antibody, or fragment thereof, capable of binding human IL-12; PGE2; LPA; NGF; CGRP; SubP; RAGE; histamine; a histamine receptor blocker; bradykinin; IL-1alpha; IL-1beta; VEGF; PLGF; methotrexate; a corticosteroid, a glucocorticoid receptor modulator; cyclosporin, rapamycin, FK506, and a non-steroidal anti-inflammatory agent.
36. The method of claim 34, wherein the disorder is an autoimmune and/or inflammatory disorder.
37. The method of claim 36, wherein the disorder is selected from the group consisting of Crohn's disease, psoriasis, plaque psoriasis, arthritis, rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis, multiple sclerosis, and ankylo sing spondylitis.
38. The method of claim 36, wherein the disorder is selected from the group consisting of a respiratory disorder; asthma; allergic and nonallergic asthma; asthma due to infection; asthma due to infection with respiratory syncytial virus (RSV); chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation; eosinophilia; fibrosis and excess mucus production; cystic fibrosis; pulmonary fibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema; allergic rhinitis; allergic enterogastritis; an inflammatory and/or autoimmune condition of the skin; an inflammatory and/or autoimmune condition of gastrointestinal organs; inflammatory bowel diseases (IBD); ulcerative colitis; Crohn's disease; an inflammatory and/or autoimmune condition of the liver; liver cirrhosis; liver fibrosis; liver fibrosis caused by hepatitis B and/or C virus; scleroderma; tumors or cancers; hepatocellular carcinoma; glioblastoma; lymphoma; Hodgkin's lymphoma; a viral infection; a bacterial infection; a parasitic infection; HTLV-1 infection; suppression of expression of protective type 1 immune responses, and suppression of expression of a protective type 1 immune response during vaccination.
39. The method of claim 35, wherein the disorder is an autoimmune and/or inflammatory disorder.
40. The method of claim 39, wherein the disorder is selected from the group consisting of Crohn's disease, psoriasis, plaque psoriasis, arthritis, rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis, multiple sclerosis, and ankylo sing spondylitis.
41. The method of claim 39, wherein the disorder is selected from the group consisting of a respiratory disorder; asthma; allergic and nonallergic asthma; asthma due to infection; asthma due to infection with respiratory syncytial virus (RSV); chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation; eosinophilia; fibrosis and excess mucus production; cystic fibrosis; pulmonary fibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema; allergic rhinitis; allergic enterogastritis; an inflammatory and/or autoimmune condition of the skin; an inflammatory and/or autoimmune condition of gastrointestinal organs; inflammatory bowel diseases (IBD); ulcerative colitis; Crohn's disease; an inflammatory and/or autoimmune condition of the liver; liver cirrhosis; liver fibrosis; liver fibrosis caused by hepatitis B and/or C virus; scleroderma; tumors or cancers; hepatocellular carcinoma; glioblastoma; lymphoma; Hodgkin's lymphoma; a viral infection; a bacterial infection; a parasitic infection; HTLV-1 infection; suppression of expression of protective type 1 immune responses, and suppression of expression of a protective type 1 immune response during vaccination.
42. A method for treating a mammal comprising administering to the mammal an effective amount of a pharmaceutical composition comprising a binding protein that binds human TNFα, the binding protein comprising at least one light chain variable region (VL region), wherein the VL region comprises the amino acid sequences of the three complementarity determining regions (CDRs) from SEQ ID NO: 1078 or the amino acid sequence of SEQ ID NO: 1078.
43. A method for reducing human TNF-α activity in a human subject suffering from a disorder in which TNF-α activity is detrimental, the method comprising administering to the human subject the binding protein of claim 40 such that human TNF-α activity in the human subject is reduced and/or treatment is achieved.
44. A method for treating a patient suffering from a disorder in which TNF-α is detrimental comprising administering to the patient the binding protein of claim 40 either before, concurrent, or after the administration to the patient of a second agent, wherein the second agent is selected from the group consisting of an antibody, or fragment thereof, capable of binding human IL-12; PGE2; LPA; NGF; CGRP; SubP; RAGE; histamine; a histamine receptor blocker; bradykinin; IL-1alpha; IL-1beta; VEGF; PLGF; methotrexate; a corticosteroid, a glucocorticoid receptor modulator; cyclosporin, rapamycin, FK506, and a non-steroidal anti-inflammatory agent.
45. The method of claim 43, wherein the disorder is an autoimmune and/or inflammatory disorder.
46. The method of claim 45, wherein the disorder is selected from the group consisting of Crohn's disease, psoriasis, plaque psoriasis, arthritis, rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis, multiple sclerosis, and ankylo sing spondylitis.
47. The method of claim 45, wherein the disorder is selected from the group consisting of a respiratory disorder; asthma; allergic and nonallergic asthma; asthma due to infection; asthma due to infection with respiratory syncytial virus (RSV); chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation; eosinophilia; fibrosis and excess mucus production; cystic fibrosis; pulmonary fibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema; allergic rhinitis; allergic enterogastritis; an inflammatory and/or autoimmune condition of the skin; an inflammatory and/or autoimmune condition of gastrointestinal organs; inflammatory bowel diseases (IBD); ulcerative colitis; Crohn's disease; an inflammatory and/or autoimmune condition of the liver; liver cirrhosis; liver fibrosis; liver fibrosis caused by hepatitis B and/or C virus; scleroderma; tumors or cancers; hepatocellular carcinoma; glioblastoma; lymphoma; Hodgkin's lymphoma; a viral infection; a bacterial infection; a parasitic infection; HTLV-1 infection; suppression of expression of protective type 1 immune responses, and suppression of expression of a protective type 1 immune response during vaccination.
48. The method of claim 44, wherein the disorder is an autoimmune and/or inflammatory disorder.
49. The method of claim 48, wherein the disorder is selected from the group consisting of Crohn's disease, psoriasis, plaque psoriasis, arthritis, rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis, multiple sclerosis, and ankylo sing spondylitis.
50. The method of claim 48, wherein the disorder is selected from the group consisting of a respiratory disorder; asthma; allergic and nonallergic asthma; asthma due to infection; asthma due to infection with respiratory syncytial virus (RSV); chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation; eosinophilia; fibrosis and excess mucus production; cystic fibrosis; pulmonary fibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema; allergic rhinitis; allergic enterogastritis; an inflammatory and/or autoimmune condition of the skin; an inflammatory and/or autoimmune condition of gastrointestinal organs; inflammatory bowel diseases (IBD); ulcerative colitis; Crohn's disease; an inflammatory and/or autoimmune condition of the liver; liver cirrhosis; liver fibrosis; liver fibrosis caused by hepatitis B and/or C virus; scleroderma; tumors or cancers; hepatocellular carcinoma; glioblastoma; lymphoma; Hodgkin's lymphoma; a viral infection; a bacterial infection; a parasitic infection; HTLV-1 infection; suppression of expression of protective type 1 immune responses, and suppression of expression of a protective type 1 immune response during vaccination.
51. The method of claim 19, wherein the binding protein:
(a) modulates a biological function of the TNF-α;
(b) neutralizes the TNF-α;
(c) diminishes the ability of the TNF-α to bind to its receptor;
(d) diminishes the ability of pro-human TNF-α, mature-human TNF-α, or truncated-human TNF-α to bind to its receptor; and/or
(e) reduces one or more of TNF-dependent cytokine production, TNF-dependent cell killing, TNF-dependent inflammation, TNF-dependent bone erosion, and TNF-dependent cartilage damage.
52. The method of claim 19, wherein the binding protein comprises:
(a) a heavy chain constant region comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 3; and
(b) a light chain constant region comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 5.
53. The method of claim 19, the at least one heavy chain variable region (VH region) comprising: (a) three CDRs from any one of SEQ ID NOs: 74-83 and 778-956; or (b) any one of SEQ ID NOs: 74-83 and 778-956.
54. The method of claim 19, the at least one light chain variable region (VL region) comprising: (a) three CDRs from any one of SEQ ID NOs: 84-93 and 957-1052; or (b) any one of SEQ ID NOs: 84-93 and 957-1052.
55. The method of claim 19, the at least one heavy chain variable region (VH region) comprising: (a) three CDRs from any one of SEQ ID NOs: 74-83, 778-956; or (b) the sequence of any one of SEQ ID NOs: 74-83 and 778-956; and the at least one light chain variable region (VL region) comprising: (c) three CDRs from any one of SEQ ID NOs: 84-93 and 957-1052; or
(d) the sequence of any one of SEQ ID NOs: 84-93 and 957-1052.
56. The method of claim 55, wherein the VH region comprises the sequence of SEQ ID NO: 74 and the VL region comprises the sequence of SEQ ID NO: 84.
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BR112014009799A2 (en) 2017-06-13
HK1200464A1 (en) 2015-08-07
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DOP2014000083A (en) 2014-06-15
JP2014534218A (en) 2014-12-18
US9803009B2 (en) 2017-10-31
TW201326207A (en) 2013-07-01
AU2012328921A1 (en) 2014-05-01
AU2012328921A8 (en) 2014-05-08
CN104093739A (en) 2014-10-08
RS20140203A1 (en) 2014-10-31
IL232129A0 (en) 2014-05-28
IN2014CN03936A (en) 2015-09-04
KR20140084253A (en) 2014-07-04
UY34409A (en) 2013-05-31
WO2013063114A1 (en) 2013-05-02
PE20141545A1 (en) 2014-11-26
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US20130164256A1 (en) 2013-06-27
ECSP14001249A (en) 2015-07-31
MX2014004981A (en) 2014-09-11
RU2014120755A (en) 2015-12-10
CL2014001054A1 (en) 2014-09-05
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WO2013063114A8 (en) 2014-03-20
CO7020873A2 (en) 2014-08-11

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