Patent Application
20220162297
The present application includes a txt sequence listing named 541262SEQLST.TXT of 171,740 bytes created Dec. 20, 2019, which is incorporated by reference.
Monoclonal antibodies as chimeric, humanized or fully human molecules have proven value as efficacious drugs for treatment of a range of medical conditions & diseases.
Antibody molecules in their native context are comprised of two heavy-chain homodimers, each of which forms a heterodimer in partnership with two identical light chain molecules (
Many diseases are comprised of multiple co-morbidities arising from multiple pathologies. Bispecific antibodies offer the ability to bind, and thereby neutralize two or more antigens per antibody molecule (
The assembly of antibody heavy chains into homodimers is driven by the CH3 domain (Gunasekaran et al. 2010). Product purity of bi-specific antibodies is dependent on two factors, heterodimeric assembly of two different heavy-chains co-expressed in the cell, and appropriate pairing of two different light chains with the respective heavy chains (
The invention provides a heterodimeric bispecific antibody (HBA) comprising a first antigen recognizing moiety (A) and a second antigen recognizing moiety (B). A contains a first light chain comprising a first light chain variable region (VL1) fused to a light chain constant region (CL), and a first heavy chain comprising a first heavy chain variable region (VH1) fused to a first heavy chain constant domain (CH1) optionally fused, through a hinge region (H), to a first heavy chain pairing partner (X). VL1 & VH1 can be either humanized, fully human, or non-human. B contains a second light chain comprising a second light chain variable region (VL2) fused to a first light chain pairing partner (m), which VL2 is different from VL1, and a second heavy chain comprising a second heavy chain variable region (VH2) fused to a second light chain pairing partner (n) fused, optionally through a hinge region (H), to a second heavy chain pairing partner (Y). VL2 and VH2 can be either humanized, fully human, or non-human. X and Y are members of a first binding pair of components from the immunoglobulin superfamily (X and Y binding pair). m and n are members of a second binding pair of components from the immunoglobulin superfamily that is different from the first binding pair (m and n binding pair). Optionally CH1 and X are fused through a constant heavy chain region (CH2) and n and Y are fused through the CH2.
In some HBA's, the CH1 is fused through H to X and n is fused through H to Y. In some HBA's the X and Y binding pair and the m and n binding pair are derived from MHC class I molecules (MHC I), MHC class II molecules (MHC II) or T-cell receptor complex of molecules (TCR). For example, the X and Y binding pair and the m and n binding pair can be any of (i) MHC II α2 domain and MHC II β2 domain, (ii) MHC I α3 domain and beta-2 microglobulin (β2M), (iii) TCR CD3 ε chain and TCR CD3 γ chain, (iv) TCR CD3 ε chain and TCR CD3 δ chain, (v) TCR antigen receptor (AgR) Cα domain and TCR AgR Cβ domain, or (vi) TCR AgR Cγ domain and TCR AgR Cδ domain. In any given pair, the actual heterologous polypeptides in the positions of X and Y or m and n can be reversed. However, X and Y are each different from both m and n.
Some HBA's of the invention comprise a first antigen recognizing moiety (C) and a second antigen recognizing moiety (D). C contains a first light chain variable region (VL1) fused to a first heavy chain variable region (VH1) fused, optionally through a hinge region (H), to a first heavy chain pairing partner (X). D comprises a second light chain variable region (VL2) fused to a second heavy chain variable region (VH2) fused, optionally through a hinge region (H), to a second heavy chain pairing partner (Y). X and Y are members of a first binding pair of components derived from the immunoglobulin superfamily (X and Y binding pair). Optionally, VH1 and X are fused through a constant heavy chain region (CH2) and VH2 and Y are fused through the CH2. In some such HBA's, the CH1 is fused through H to X and n is fused through H to Y. In some HBA's the X and Y binding pair and the m and n binding pair are derived from MHC class I molecules (MHC I), MHC II or TCR.
Any of the HBA's can include a CH2, such that VH1 and X are fused through a constant heavy chain region (CH2) and VH2 and Y are fused through CH2. In addition, or alternatively, X is fused to a third heavy chain binding partner (p) and Y is fused to a fourth heavy chain binding partner (q). If present, p and q are members of a third binding pair of components derived from the immunoglobulin superfamily (p and q binding pair). In some HBA's the p and q binding pair is derived from MHC I, MHC II or TCR. p and q can be the same as X and Y, respectively, or the p and q binding pair can be different from the X and Y binding pair. However, if a p and q binding pair and an m and n binding pair are both present, then p and q are each different from both m and n.
For example, if the X and Y binding pair is MHC I α3 domain and beta-2 microglobulin (β2M), (a) the m and n binding pair, if present, can be MHC II α2 domain and MHC II β2 domain, TCR CD3 ε chain and TCR CD3 γ chain, TCR CD3 ε chain and TCR CD3 δ chain, TCR AgR Cα domain and TCR AgR Cβ domain or TCR AgR Cγ domain and TCR AgR Cδ domain; and (b) the p and q binding pair can be MHC II α2 domain and MHC II β2 domain, TCR CD3 ε chain and TCR CD3 γ chain, TCR CD3 ε chain and TCR CD3 δ chain, TCR AgR Cα domain and TCR AgR Cβ domain or TCR AgR Cγ domain and TCR AgR Cδ domain, but only if different from the pair in (a).
In some of the HBA's, CL is human Cκ or Cλ. Some heavy chain segments, such as, for example, CH1, H and CH2 are from human IgA, IgD, IgM, IgE, or IgG isotypes 1, 2, 3 or 4, and can be, for example, from IgG1.
The HBA's can include at least one of A, B, C or D derived from a therapeutic antibody, such as any of the therapeutic antibodies disclosed herein, for example, A or B, but not both, can be derived from adalimumab. Some HBA's contain VH1, CH1, VL1, CL from adalimumab, X is MHC I α3 domain and Y is beta-2 microglobulin (β2M). As discussed above, the specific heterologous polypeptides in positions X and Y can be reversed. As provided in further detail infra, various substitutions of adalimumab CH3 with pairing partners from the immunoglobulin superfamily (IgSF) can readily be made with the teachings herein.
Also provided herein are polynucleotides comprising one or more nucleic acid(s) encoding the first heavy chain, the second light chain or the second heavy chain discussed supra. Also provided are polynucleotides comprising one or more nucleic acid(s) encoding moiety C or moiety D discussed supra. Vectors comprising one or more of these polynucleotides are provided, as well as host cells containing such polynucleotides or vectors. The invention also provides methods of making HBA's comprising culturing such host cells under conditions so as to express the nucleic acid(s) encoding the HBA, and recovering the HBA.
The invention further provides a heterodimeric bispecific fusion protein (HBFP) comprising a first (R1) comprising a first biologically active moiety (R1) and a second moiety (R2) comprising a second biologically active moiety (R2), which are fused to heterologous polypeptides in positions X and Y as discussed supra.
The invention further provides a method for treating a patient having or at risk for having cancer, an autoimmune or inflammatory condition, a neurodegenerative disease, an infectious disease, osteoporosis, dyslipidemia, macular degeneration, a blood coagulation disorder, a cardiovascular disease or disorder, an organ transplant, diabetes, influenza, a muscle wasting disorder or a gastrointestinal disease or disorder, comprising administering to the patient a therapeutically effective dose of any of the HBA's or HBFP's described herein.
Some patients have cancer, for example, ovarian cancer, prostate cancer, breast cancer, colorectal cancer, non-small cell lung caracinoma, gastrointestinal cancer, hematological cancer, metastatic cancer, squamous cell carcinoma, head and neck cancer, solid tumors, glioblastoma, neuroblastoma, testicular cancer, adrenocortical carcinoma, melanoma, non-Hodgkin's lymphoma or pancreatic cancer. Some patients have a hematological cancer such as lymphoma, non-Hodkin's lymphoma, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, or Hodgkin's lymphoma.
Some patients have an autoimmune or inflammatory condition, such as inflammatory bowl disease, ulcerative colitis, Crohn's disease, rheumatoid arthritis, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, multiple sclerosis, systematic lupus erythematosus or uveitis.
Some patients have a neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease or ALS.
α domain in uppercase italic and wavy underline, the TCR Cα domain in uppercase bold, the juxta-membrane connecting peptide between the TCR Cα domain and the trans-membrane domain in uppercase dotted underline.
β domain in uppercase italic and wavy underline, the TCR Cβ domain in uppercase bold, the juxta-membrane connecting peptide between the TCR Cβ domain and the trans-membrane domain in uppercase dotted underline.
γ domain in uppercase italic and wavy underline, the TCR Cγ domain in uppercase bold, the juxta-membrane connecting peptide between the TCR Cγ domain and the trans-membrane domain in uppercase dotted underline.
δ domain in uppercase italic and wavy underline, the TCR Cδ domain in uppercase bold, the juxta-membrane connecting peptide between the TCR Cδ domain and the trans-membrane domain in uppercase dotted underline.
Note: The typefaces associated with each sequence have the same meaning as indicated for
SEQ ID NO:1 sets forth a nucleic acid sequence encoding the adalimumab light chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:2 sets forth the amino acid sequence of the adalimumab light chain (leader peptide inclusive).
SEQ ID NO:3 sets forth a nucleic acid sequence encoding the adalimumab heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:4 sets forth the amino acid sequence of the adalimumab heavy chain (leader peptide inclusive).
SEQ ID NO:5 sets forth a nucleic acid sequence encoding an exemplary adalimumab MHC-Iα3 hybrid heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:6 sets forth the amino acid sequence of an exemplary adalimumab MHC-Iα3 hybrid heavy chain (leader peptide inclusive).
SEQ ID NO:7 sets forth a nucleic acid sequence encoding an MHC I HLA-A (A*68 allele) α3 domain (stop codon inclusive).
SEQ ID NO:8 sets forth the amino acid sequence of an MHC I HLA-A (A*68 allele) α3 domain.
SEQ ID NO:9 sets forth a nucleic acid sequence encoding an exemplary adalimumab β2 microglobulin hybrid heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:10 sets forth the amino acid sequence of an exemplary adalimumab β2 microglobulin hybrid heavy chain (leader peptide inclusive).
SEQ ID NO:11 sets forth a nucleic acid sequence encoding a β2 microglobulin (stop codon inclusive).
SEQ ID NO:12 sets forth the amino acid sequence of a β2 microglobulin.
SEQ ID NO:13 sets forth a nucleic acid sequence encoding an exemplary adalimumab MHC II α2 domain (derived from HLA-DR alpha) hybrid heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:14 sets forth the amino acid sequence of an exemplary adalimumab MHC II α2 domain (derived from HLA-DR alpha) hybrid heavy chain (leader peptide inclusive).
SEQ ID NO:15 sets forth a nucleic acid sequence encoding an MHC II α2 domain from HLA-DR alpha (stop codon inclusive).
SEQ ID NO:16 sets forth the amino acid sequence of an MHC II α2 domain from HLA-DR alpha.
SEQ ID NO:17 sets forth a nucleic acid sequence encoding an exemplary adalimumab MHC II β2 domain (derived from HLA-DR beta1) hybrid heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:18 sets forth the amino acid sequence of an exemplary adalimumab MHC II β2 domain (derived from HLA-DR beta1) hybrid heavy chain (leader peptide inclusive).
SEQ ID NO:19 sets forth a nucleic acid sequence encoding an MHC II β2 domain from HLA-DR beta1 (stop codon inclusive).
SEQ ID NO:20 sets forth the amino acid sequence of an MHC II β2 domain from HLA-DR beta1.
SEQ ID NO:21 sets forth a nucleic acid sequence encoding an exemplary adalimumab TCR α domain hybrid heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:22 sets forth the amino acid sequence of an exemplary adalimumab TCR α domain hybrid heavy chain (leader peptide inclusive).
SEQ ID NO:23 sets forth a nucleic acid sequence encoding a TCR α domain (stop codon inclusive).
SEQ ID NO:24 sets forth the amino acid sequence of a TCRα domain.
SEQ ID NO:25 sets forth a nucleic acid sequence encoding an exemplary adalimumab TCR β domain hybrid heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:26 sets forth the amino acid sequence of an exemplary adalimumab TCR β domain hybrid heavy chain (leader peptide inclusive).
SEQ ID NO:27 sets forth a nucleic acid sequence encoding a TCR β domain (stop codon inclusive).
SEQ ID NO:28 sets forth the amino acid sequence of a TCR β domain.
SEQ ID NO:29 sets forth a nucleic acid sequence encoding an exemplary adalimumab TCR γ domain hybrid heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:30 sets forth the amino acid sequence of an exemplary adalimumab TCR γ domain hybrid heavy chain (leader peptide inclusive).
SEQ ID NO:31 sets forth a nucleic acid sequence encoding a TCR γ domain (stop codon inclusive).
SEQ ID NO:32 sets forth the amino acid sequence of a TCRγ domain.
SEQ ID NO:33 sets forth a nucleic acid sequence encoding an exemplary adalimumab TCR δ domain hybrid heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:34 sets forth the amino acid sequence of an exemplary adalimumab TCR δ domain hybrid heavy chain (leader peptide inclusive).
SEQ ID NO:35 sets forth a nucleic acid sequence encoding a TCR δ domain (stop codon inclusive).
SEQ ID NO:36 sets forth the amino acid sequence of a TCRδ domain.
SEQ ID NO:37 sets forth a nucleic acid sequence encoding an exemplary adalimumab CD3 ε-chain ecto-domain hybrid heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:38 sets forth the amino acid sequence of an exemplary adalimumab CD3 ε-chain ecto-domain hybrid heavy chain (leader peptide inclusive).
SEQ ID NO:39 sets forth a nucleic acid sequence encoding a CD3 ε-chain ecto-domain (stop codon inclusive).
SEQ ID NO:40 sets forth the amino acid sequence of a CD3 ε-chain ecto-domain.
SEQ ID NO:41 sets forth a nucleic acid sequence encoding an exemplary adalimumab CD3 γ-chain ecto-domain hybrid heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:42 sets forth the amino acid sequence of an exemplary adalimumab CD3 γ-chain ecto-domain hybrid heavy chain (leader peptide inclusive).
SEQ ID NO:43 sets forth a nucleic acid sequence encoding a CD3 γ-chain ecto-domain (stop codon inclusive).
SEQ ID NO:44 sets forth the amino acid sequence of a CD3 γ-chain ecto-domain.
SEQ ID NO:45 sets forth a nucleic acid sequence encoding an exemplary adalimumab CD3 δ-chain ecto-domain hybrid heavy chain (start codon, leader peptide and stop codon inclusive).
SEQ ID NO:46 sets forth the amino acid sequence of an exemplary adalimumab CD3 δ-chain ecto-domain hybrid heavy chain (leader peptide inclusive).
SEQ ID NO:47 sets forth a nucleic acid sequence encoding a CD3 δ-chain ecto-domain (stop codon inclusive).
SEQ ID NO:48 sets forth the amino acid sequence of a CD3 δ-chain ecto-domain.
SEQ ID NO:49 sets forth the nucleic acid sequence of an exemplary CH1 domain.
SEQ ID NO:50 sets forth the amino acid sequence of an exemplary CH1 domain.
SEQ ID NO:51 sets forth the nucleic acid sequence of an exemplary hinge region.
SEQ ID NO:52 sets forth the amino acid sequence of an exemplary hinge region.
SEQ ID NO:53 sets forth the nucleic acid sequence of an exemplary CH2 domain.
SEQ ID NO:54 sets forth the amino acid sequence of an exemplary CH2 domain.
SEQ ID NO:55 sets forth the nucleic acid sequence of an exemplary CH3 domain.
SEQ ID NO:56 sets forth the amino acid sequence of an exemplary CH3 domain.
SEQ ID NO:57 sets forth a nucleic acid sequence encoding an exemplary adalimumab MHC-I HLA-A (A*68 allele) α3 hybrid heavy chain.
SEQ ID NO:58 sets forth the amino acid sequence of an exemplary adalimumab MHC-I HLA-A (A*68 allele) α3 hybrid heavy chain.
SEQ ID NO:59 sets forth a nucleic acid sequence encoding an exemplary adalimumab β2 microglobulin hybrid heavy chain.
SEQ ID NO:60 sets forth the amino acid sequence of an exemplary adalimumab β2 microglobulin hybrid heavy chain.
SEQ ID NO:61 sets forth a nucleic acid sequence encoding an exemplary adalimumab MHC II α2 domain (from HLA-DR alpha) hybrid heavy chain.
SEQ ID NO:62 sets forth the amino acid sequence of an exemplary adalimumab MHC II α2 domain (from HLA-DR alpha) hybrid heavy chain.
SEQ ID NO:63 sets forth a nucleic acid sequence encoding an exemplary adalimumab MHC II β2 domain (from HLA-DR beta1) hybrid heavy chain.
SEQ ID NO:64 sets forth the amino acid sequence of an exemplary adalimumab MHC II β2 domain (from HLA-DR beta1) hybrid heavy chain.
SEQ ID NO:65 sets forth a nucleic acid sequence encoding an exemplary adalimumab TCR α domain hybrid heavy chain.
SEQ ID NO:66 sets forth the amino acid sequence of an exemplary adalimumab TCR α domain hybrid heavy chain.
SEQ ID NO:67 sets forth a nucleic acid sequence encoding an exemplary adalimumab TCR β domain hybrid heavy chain.
SEQ ID NO:68 sets forth the amino acid sequence of an exemplary adalimumab TCR β domain hybrid heavy chain.
SEQ ID NO:69 sets forth a nucleic acid sequence encoding an exemplary adalimumab TCR γ domain hybrid heavy chain.
SEQ ID NO:70 sets forth the amino acid sequence of an exemplary adalimumab TCR γ domain hybrid heavy chain.
SEQ ID NO:71 sets forth a nucleic acid sequence encoding an exemplary adalimumab TCR δ domain hybrid heavy chain.
SEQ ID NO:72 sets forth the amino acid sequence of an exemplary adalimumab TCR δ domain hybrid heavy chain.
SEQ ID NO:73 sets forth a nucleic acid sequence encoding an exemplary adalimumab CD3 ε-chain ecto-domain hybrid heavy chain.
SEQ ID NO:74 sets forth the amino acid sequence of an exemplary adalimumab CD3 ε-chain ecto-domain hybrid heavy chain.
SEQ ID NO:75 sets forth a nucleic acid sequence encoding an exemplary adalimumab CD3 γ-chain ecto-domain hybrid heavy chain.
SEQ ID NO:76 sets forth the amino acid sequence of an exemplary adalimumab CD3 γ-chain ecto-domain hybrid heavy chain.
SEQ ID NO:77 sets forth a nucleic acid sequence encoding an exemplary adalimumab CD3 δ-chain ecto-domain hybrid heavy chain.
SEQ ID NO:78 sets forth the amino acid sequence of an exemplary adalimumab CD3 δ-chain ecto-domain hybrid heavy chain.
SEQ ID NO:79 sets forth a nucleic acid sequence encoding an MHC I HLA-A (A*68 allele) α3 domain.
SEQ ID NO:80 sets forth a nucleic acid sequence encoding a β2 microglobulin.
SEQ ID NO:81 sets forth a nucleic acid sequence encoding an MHC II α2 domain from HLA-DR alpha.
SEQ ID NO:82 sets forth a nucleic acid sequence encoding an MHC II β2 domain from HLA-DR beta1.
SEQ ID NO:83 sets forth a nucleic acid sequence encoding a TCR α domain.
SEQ ID NO:84 sets forth a nucleic acid sequence encoding a TCR β domain.
SEQ ID NO:85 sets forth a nucleic acid sequence encoding a TCR γ domain.
SEQ ID NO:86 sets forth a nucleic acid sequence encoding a TCR δ domain.
SEQ ID NO:87 sets forth a nucleic acid sequence encoding a CD3 ε-chain ecto-domain.
SEQ ID NO:88 sets forth a nucleic acid sequence encoding a CD3 γ-chain ecto-domain.
SEQ ID NO:89 sets forth a nucleic acid sequence encoding a CD3 δ-chain ecto-domain.
The heterodimeric bispecific antibodies (HBA's) or heterodimeric bispecific Fc fusion proteins (HBFP's) are typically provided in isolated form. This means that an HBA or HBFP is typically at least 50% w/w pure of interfering proteins and other contaminants arising from its production or purification but does not exclude the possibility that the HBA or HBFP is combined with an excess of pharmaceutical acceptable carrier(s) or other vehicle intended to facilitate its use. Sometimes HBA's or HBFP's are at least 60, 70, 80, 90, 95 or 99% w/w pure of interfering proteins and contaminants from production or purification. Often an HBA or HBFP is the predominant macromolecular species remaining after its purification.
Specific binding of an HBA or HBFP to its target antigen means an affinity of at least 106, 107, 108, 109, or 1010 M−1. Specific binding is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit (e.g., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not however necessarily imply that an HBA or HBFP binds one and only one target.
A basic antibody structural unit is a tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region means a light chain variable region without the light chain signal peptide. However, reference to a variable region does not mean that a signal sequence is necessarily present; and in fact signal sequences are cleaved once the HBA's of the invention have been expressed and secreted. A pair of heavy and light chain variable regions, or heavy chain variable region alone as in the case of Camelids or engineered mimetics thereof, defines a binding region of an antibody. The carboxy-terminal portion of the light and heavy chains respectively defines light and heavy chain constant regions. The heavy chain constant region is primarily responsible for effector function. In IgG antibodies, the heavy chain constant region is divided into CH1, hinge, CH2, and CH3 regions. The CH1 region binds to the light chain constant region by disulfide and noncovalent bonding. The hinge region provides flexibility between the binding and effector regions of an antibody and also provides sites for intermolecular disulfide bonding between the two heavy chain constant regions in a tetramer subunit. The CH2 and CH3 regions are the primary site of effector functions and FcRn binding. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” segment of about 12 or more amino acids, with the heavy chain also including a “D” segment of about 10 or more amino acids. (See generally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7) (incorporated by reference in its entirety for all purposes).
The mature variable regions of each light/heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites, i.e., is bivalent. In natural antibodies, the binding sites are the same. However, bispecific antibodies can be made in which the two binding sites are different (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). The variable regions all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chain variable regions or between different light chain variable regions are assigned the same number. Although Kabat numbering can be used for antibody constant regions, the EU index is more commonly used, as is the case in this application.
In a mono-specific bivalent antibody, the two heavy chain and two light chain variable regions are the same. In a bispecific bivalent antibody, there are two different heavy and light chain variable region pairings or “arms” with different binding specificities.
The term “antibody” includes any form of antibody with at least one binding region including monovalent fragments, bivalent tetrameric units of two heavy chains and light chains, and higher order complexes, particularly trimers, tetramers and pentamers of bivalent units. An antibody can be mono-specific in which case all binding regions have the same specificity or multi-specific in which the binding sites have at least two specificities.
The term “epitope” refers to a site on an antigen to which an antibody or HBA binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. Some antibodies bind to an end-specific epitope, meaning an antibody binds preferentially to a polypeptide with a free end relative to the same polypeptide fused to another polypeptide resulting in loss of the free end. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography, cryo-electron microscopy, and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).
The term “antigen” or “target antigen” indicates a target molecule bound by an antibody. An antigen may be a protein of any length (natural, synthetic or recombinantly expressed), a nucleic acid or carbohydrate among other molecules. Antigens include receptors, ligands, counter receptors, and coat proteins.
A heterologous polypeptide in a fusion protein is a polypeptide not naturally linked to an immunoglobulin constant region. Such a polypeptide can be a full-length protein or any fragment thereof of sufficient length to retain specific binding to the antigen or ligand bound by the full-length protein. For example, a heterologous polypeptide can be a receptor extracellular domain or ligand thereto.
The term “fused” when used in describing the structure of heterodimeric bispecific antibodies or heterodimeric bispecific fusion proteins means that the segments in question are either directly connected or connected through intervening amino acids.
Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can also be defined X-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibits binding of the reference antibody by at least 50% but preferably 75%, 90% or 99% as measured in a competitive binding assay. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.
The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another.
Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention for a variable region or EU numbering for a constant region. For other proteins, sequence identity can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or BLAST (basic local alignment search tool) as defined in the national center for biotechnology information web site world wide web blast.ncbi.nlm.nih.gov/Blast.cgi, using default gap parameters, or by inspection, and the best alignment. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.
Compositions or methods “comprising” one or more recited elements may include other elements not specifically recited. For example, a composition that comprises antibody may contain the antibody alone or in combination with other ingredients.
A humanized antibody is a genetically engineered antibody in which the CDRs from a non-human “donor” antibody are grafted into human “acceptor” antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539, Carter, U.S. Pat. No. 6,407,213, Adair, U.S. Pat. No. 5,859,205 6,881,557, Foote, U.S. Pat. No. 6,881,557). The acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having some or all CDRs entirely or substantially from a donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences. Similarly a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, substantially from human heavy chain variable region framework and constant region sequences. Similarly a humanized light chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences. Other than nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least 85%, 90%, 95% or 100% of corresponding residues (as defined by Kabat) are identical between the respective CDRs. The variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially from a human variable region framework sequence or human constant region respectively when at least 85, 90, 95 or 100% of corresponding residues defined by Kabat are identical.
Although humanized antibodies often incorporate all six CDRs (preferably as defined by Kabat) from a mouse antibody, they can also be made with less than all CDRs (e.g., at least 3, 4, or 5 CDRs from a mouse antibody) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164:1432-1441, 2000).
A chimeric antibody is an antibody in which the mature variable regions of light and heavy chains of a non-human antibody (e.g., a mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or entirely retain the binding specificity of the mouse antibody, and are about two-thirds human sequence.
A veneered antibody is a type of humanized antibody that retains some and usually all of the CDRs and some of the non-human variable region framework residues of a non-human antibody but replaces other variable region framework residues that may contribute to B- or T-cell epitopes, for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) with residues from the corresponding positions of a human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially from a non-human antibody and the variable region frameworks of the non-human antibody are made more human-like by the substitutions.
A human antibody can be isolated from a human, or otherwise result from expression of human immunoglobulin genes (e.g., in a transgenic mouse, in vitro or by phage display). Methods for producing human antibodies include the trioma method of Oestberg et al., Cys muoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, use of transgenic mice including human immunoglobulin genes (see, e.g., Lonberg et al., WO93/12227 (1993); U.S. Pat. Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991) and phage display methods (see, e.g. Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, U.S. Pat. Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743 and 5,565,332.
The invention provides bispecific heterodimeric antibodies with modified heavy chain IgG constant regions that promote efficient assembly of antibody heavy chain heterodimer pairs, as well as arm specific pairing of heavy and light chains. Some antibodies of the invention have modified light chain constant regions that promote arm specific pairing of the modified light chain with the desired heavy chain for efficient assembly of antibody light chain+heavy chain heterodimer pairs. A heterodimeric bispecific antibody as described herein comprises two polypeptide chains having different amino acid sequences, which, together, can bind to two different antigens, or two different epitopes on the same antigen.
The heterodimeric antibodies include heterologous components derived from polypeptides or proteins, e.g. the immunoglobulin superfamily (IgSF), that naturally occur as heterodimers, or are engineered to assemble as heterodimers during expression in cells or in-vitro. As used herein, the term “IgSF” excludes antibody domains. Domains from the IgSF in place of CH3 domain and/or the CH1+CL domain of the antibody heavy and light chains mediate the correct assembly of the bispecific antibodies, thereby reducing mispaired contaminants and enhancing overall product purity and yield.
The heterodimeric bispecific antibodies of the invention (HBA's) specifically bind to different targets. The targets can be completely different target proteins or antigens, or can be different epitopes within a common target protein or antigen. Thus, the HBA comprises a first antigen recognizing moiety (A) and a second antigen recognizing moiety (B), where A and B bind to different target proteins or antigens or different epitopes within a common target protein or antigen.
The first antigen recognizing moiety (A) has a light chain variable region (VL1) fused to a light chain constant region (CL). VL1 can be either humanized, fully human, or non-human. A also has a heavy chain variable region (VH1) fused to a heavy chain constant domain (CH1), which is fused to a heterologous molecule in place of CH3 (X). The term “fused” VH1 can be either humanized, fully human, or non-human. The heterologous polypeptide is derived from a component within the immunoglobulin superfamily (IgSF) that can form heterodimers with at least one other component from the IgSF (pairing partner). In some HBA's of the invention, the CH1 is fused to X through an antibody hinge region (H). In some HBA's, CH1 and X are fused through constant heavy chain region CH2 and in some HBA's, CH1 is fused to CH2 through H. CL can be Cκ light chain constant region or Cλ, light chain constant region. CH1, H and CH2 can be from any of the heavy chain constant region isotypes, such as, for example, human IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 and IgG4. In some HBA's VL1 is fused directly to VH1.
The second antigen recognizing moiety (B) has a light chain variable region (VL2), which is different from VL1. VL2 can be either humanized, fully human, or non-human. To promote appropriate pairing of the light and heavy chains of B, in place of CL, VL2 is fused to a heterologous polypeptide (m) in place of CL. This heterologous polypeptide is derived from the IgSF that is not X or the pairing partner for X. B also has a heavy chain variable region (VH2), which is different from VH1. VH2 can be either humanized, fully human, or non-human. VH2 is fused to heterologous polypeptide in place of CH1 (n). n is derived from the IgSF pairing partner form, and is fused to the IgSF pairing partner for X in place of CH3 (Y). In some HBA's n is fused to Y through CH2. In some HBA's, n is fused to Y through H and in some HBA's n is fused to Y through H and CH2. To effect optimal assembly of the antibody heavy and light chains, m and n of the m/n binding pair are different from both X and Y of the X/Y binding pair in any given HBA. In some HBA's, VL2 is fused directly to VH2.
In some HBA's, the CL, CH1 and CH2 are derived from the corresponding mono-specific antibody from which the HBA is derived.
Some of the heterodimeric antibodies of the invention involve a replacement of the homo-dimer promoting CH3 domain with protein domains that adopt the canonical immunoglobulin fold (Bork et al. 1994; Smith and Xue 1997; Barclay 2003) and have the same pfam identifier as native CH3. Table 1 lists a set of representative naturally occurring heterodimers in the immunoglobulin superfamily (IgSF) that meet these criteria.
Thus, the X/Y binding pair can be derived from any of a number of IgSF heterodimers meeting the above criteria, including MHC class I molecules (MHC I), MHC class II molecules (MHC II) and T-Cell receptor complex of molecules (TCR).
The domain swap of CH3 in antibody heavy chain with domains from molecules that naturally exist as heterodimer pairs drives assembly of heavy chain heterodimers for the production of bi-specific antibodies, as illustrated in
Thus, in addition to α3 domain from any of the MHC-I haplotypes listed in Table 2, one may substitute the α3 domain from the cognate MHC-I heavy chains listed in Table 2 to equal or better effect in promoting heterodimerization of antibody heavy chains.
Four basic formats comprised of heterodimer heavy chains can be envisioned as illustrated schematically in
The approach illustrated schematically in λ) domain on the light chain of an arm are replaced by heterodimer forming domains from, for example, the molecules listed in Tables 1 and 2. For example, a paired domain swap of CH1 domain on one heavy chain and the Cκ domain of the corresponding light chain with cognate domains from a different set of heterodimer promoting domains (an m/n pair) are used to effect “arm-specific” pairing of light chains with heterodimer heavy chains (
Tables 3-9 list some combinatorial solutions for domain matching of any given X/Y pair in place of CH3 on the heavy chains with potential m/n pairs in place of CH1 and CL (Cκ or Cλ) domains on the heavy chain and light chain, respectively. For proper assembly, m and n are different from X and Y. Some heterodimeric bispecific antibodies may additionally include an extended domain p/q as shown in
As indicated in Table 3, for any given binding pair, the specific positions can be reversed. For example, Table 4 is not limited to the specific positions within each binding pair shown. Table 4 is intended to encompass HBA's in which X is MHC I (or homolog) α3 domain and Y is β2m, and in which m is MHC II β2 domain and n is MHC II α2 domain or m is TCR AgR Cβ domain and n is TCR AgR Cα domain or m is TCR AgR Cδ domain and n is TCR AgR Cγ domain or m is TCR CD3 γ-chain and n is TCR CD3 ε-chain or m is TCR CD3 δ-chain and n is TCR CD3 ε-chain. The same rationale for selecting from among the various possible permutations applies for each of the HBA's of the inventions, including those listed in any of Tables 3-10.
In some HBA's, when the X/Y binding pair is derived from MHC II, the m/n binding pair is derived from MHC I or TCR. Some X/Y binding pairs include MHC II α2 domain and MHC II β2 domain. In some such HBA's, X is MHC II α2 domain and Y is MHC II β2 domain or X and Y are MHC II β2 domain and MHC II α2 domain, respectively, and m and n can be MHC I α3 domain and β2M, respectively, or β2M and MHC I α3 domain, respectively. For example, in some HBA's, X is MHC II α2 domain, Y is MHC II β2 domain, m is MHC I α3 domain and n is β2M. In other HBA's, X is MHC II β2 domain, Y is MHC II α2 domain, m is β2M and n is MHC I α3 domain. The same rationale can be applied to select for the HBA's of the invention the X/Y binding pairs and m/n binding pairs from among the various possible permutations of IgSF pairing partners, including those listed in any of Tables 3-10.
In some HBA's, when the X/Y binding pair is derived from TCR, the m/n binding pair is derived from MHC I, MHC II or TCR. The X/Y binding pair can be TCR AgR Cα domain and TCR AgR Cβ domain and the m/n binding pair can be selected from the m and n pairing partners listed in Table 6.
The X/Y binding pair of some HBA's is TCR AgR Cγ domain and TCR AgR Cδ domain and the m/n binding pair can be selected from the m and n pairing partners listed in Table 7.
For some HBA's, the X/Y binding pair is TCR CD3 ε chain and TCR CD3 γ chain and the m/n binding pair can be selected from the m and n pairing partners listed in Table 8.
The X/Y binding pair for some HBA's is TCR CD3 ε chain and TCR CD3 δ chain and the m/n binding pair can be selected from the m and n pairing partners listed in Table 9.
Another aspect of this invention encompasses an extended format heterodimeric heavy chain in which the X/Y binding pair is coupled with a p/q binding pair in a tandem manner on the heavy chain, as illustrated in
chain
Following the guidance provided supra for selecting pairing partners for positions within an HBA, the amino acid sequences for the MHC I α3/β2 microglobulin, β2 microglobulin/MHC I α3, MHC II α2/MHC II β2, MHC II β2/MHC II α2, TCR α/TCR MB, TCR
β/TCR
α, TCR
γ/TCR
δ, TCR
δ/TCR
γ, CD3 ε-chain/CD3 γ-chain, CD3 γ-chain/CD3 ε-chain, CD3 ε-chain/CD3 δ-chain and CD3 δ-chain/CD3 ε-chain binding pairs for positions X/Y, p/q and/or m/n can be selected, for example, from SEQ ID NO:7/11, 11/7, 15/19, 19/15, 23/27, 27/23, 31/35, 35/31, 39/43 and 43/39, respectively.
An alternate solution to the problem of light chain pairing is envisioned wherein the two different heavy chains are expressed such that the amino-terminus of the variable region of each heavy chain is covalently linked to the carboxy-terminus of the corresponding light chain to form a single chain Fv (where each Fv recognizes a different antigen, or different epitope on the same antigen), in the context of CH3 domain swapped heterodimer promoting domains. In such HBA's, the one antigen recognizing moiety has a light chain variable region (VL1) fused to a heavy chain variable region (VH1), which is fused to a heterologous polypeptide derived from the IgSF (X). In some HBA's VH1 is fused through a hinge region (H) to X. The other antigen recognizing moiety has a different light chain variable region (VL2) fused to a different heavy chain variable region (VH2), which is fused to the pairing partner for X in the IgSF (Y). In some HBA's, VH2 is fused through H to Y. Some of the HBA's include a CH2 region, through which VH1 is fused to X and VH2 is fused to Y. In some HBA's VH1 is fused to X through H and CH2, and VH2 is fused to Y through H and CH2. Some examples of such formats are illustrated schematically in
Structural information can be used to guide the design of CH3 domain swapped heavy chain constructs. The pdb identifiers of some of the reference crystal structures are listed in Table 1. Visual inspection of the respective structures enables demarcation of the respective Ig C-1 pfam set domains in each representative member of the IgSF selected for this purpose. Some ribbon diagrams may be created for this purpose, for example, by using for example Macpymol, Cn3D, or similar structure modeling and/or visualization programs.
In order to maximize proper folding and assembly of the heterodimer forming domains in the context of an Ig heavy chain, i.e. VH1-CH1-H-CH2-X+VH2-CH1-H-CH2-Y where X and Y are, for example, as defined in Table 1, some heterodimeric bispecific antibodies retain the flexible linker separating CH2 and CH3 domains in the Ig heavy chain as a carboxy terminal extension at the end of CH2 domain. Some such heterodimeric bispecific antibodies also retain the analogous flexible loop region separating the selected heterodimer forming domains from the immediately upstream domain in the parent molecule, for fusion to the flexible linker carboxy-terminal to the heavy chain CH2 domain.
For example, some adalimumab hybrid heavy chains in which the adalimumab CH3 domain is replaced with MHC-I α3 domain, β2 microglobulin, MHC II α2 domain, MHC II β2 domain, TCR α domain, TCR
β domain, TCR
γ domain, TCR E
δ domain, CD3 ε-chain ecto-domain, CD3 γ-chain ecto-domain and CD3 δ-chain ecto-domain have the amino acid sequence of SEQ ID NO's: 6 or 58, 10 or 60, 14 or 62, 18 or 64, 22 or 66, 26 or 68, 30 or 70, 34 or 72, 38 or 74, 42 or 76 and 46 or 78, respectively.
The strategies detailed supra can also be applied for expression and manufacturing heterodimeric bispecific fusion proteins (HBFP's), as illustrated in
The bispecific fusion proteins of the invention enjoy a prolonged half-life compared to individual proteins or fusion proteins lacking features endowed by the Fc region of antibodies
R1 and R2 can be any of a number of therapeutic proteins, including ligand binding extracellular domains of cell surface receptors for disease exacerbating ligands or enzymes. A list of commercially approved FDA Fc-fusion proteins, and Fc fusion proteins in clinical development is provided in tables 11 and 12, respectively.
The invention further provides nucleic acids encoding any of the heavy and light chains described above, for example, X, Y, p, q, m and n. Included are polynucleotides having one or more nucleic acids that encode the heavy chain and light chain of antigen recognizing moiety A, for example, a hybrid derived from adalimumab or any antibody with therapeutic potential such as an therapeutic antibody disclosed infra, the light chain of antigen recognizing moiety B and the heavy chain of antigen recognizing moiety B, for example a hybrid derived from a second antibody with therapeutic potential such as an antibody disclosed infra. Some polynucleotides encode one or more of the antigen recognizing moieties of the Fv-like HBA's or the HBFP's discussed above.
One can readily combine the nucleic acid sequences of the applicable segments of known or new therapeutic antibodies with nucleic acid sequences encoding the IgSF pairing partners of choice. Following the guidance provided supra for selecting pairing partners for positions within an HBA, nucleic acid sequences encoding the MHC I α3/β2 microglobulin, β2 microglobulin/MHC I α3, MHC II α2/MHC II β2, MHC II β2/MHC II α2, TCR α/TCR
β, TCR
β/TCR
α, TCR
γ/TCR
δ, TCR
δ/TCR
γ, CD3 ε-chain/CD3 γ-chain, CD3 γ-chain/CD3 ε-chain, CD3 ε-chain/CD3 δ-chain and CD3 δ-chain/CD3 ε-chain binding pairs for positions X/Y, p/q and/or m/n can be selected, for example, from SEQ ID NO:7/11, 11/7, 79/80, 80/79, 15/19, 19/15, 81/82, 82/81, 23/27, 27/23, 83/84, 84/83, 31/35, 35/31, 85/86, 86/85, 39/43, 87/88, 88/87, and 43/39, 87/89, 89/87, respectively.
For example, some adalimumab hybrid heavy chains in which the adalimumab CH3 domain is replaced with MHC-I α3 domain, β2 microglobulin, MHC II α2 domain, MHC II β2 domain, TCR α domain, TCR
β domain, TCR
γ domain, TCR
δ domain, CD3 ε-chain ecto-domain, CD3 γ-chain ecto-domain and CD3 δ-chain ecto-domain are encoded by the nucleic acid sequences of SEQ ID NO's: 5 or 57, 9 or 59, 13 or 61, 17 or 63, 21 or 65, 25 or 67, 29 or 69, 33 or 71, 37 or 73, 41 or 75 and 45 or 77, respectively.
HBA's incorporating the heavy chain modifications described above can be made to any target molecule. The HBA's are particularly useful for surface-bound or circulating target proteins (e.g., on cells or viruses) in which aggregation or neutralization of the target protein induces a desired response. The desired response can be, for example, clearing of a target protein, reducing aggregation of a target protein, clearing of a cell or virus bearing a target, signal transduction through a receptor, e.g., inducing apoptosis or cytostasis, inhibiting a receptor binding to a ligand or counterreceptor, or internalization of an HBA conjugated to a toxic agent. HBA's can be made to the same targets as existing therapeutic antibodies or fusion proteins or can be derivatized versions of therapeutic antibodies or fusion proteins in which one or more of the existing constant regions have been replaced by the heterologous polypeptides of the present invention.
Thus, the teachings herein can be applied to create novel HBA's using mono-specific antibodies having properties of interest, such as, for example, antibodies with established therapeutic efficacy. For example, the variable regions of A or B or both can be derived from abagoavomab, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, afutuzumab, alacixumab pegol, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafeatox, anetumab ravtansine, anifrolumab, anrukinzumab, apolizumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atizumab, atorolimumab, avelumab, bapeineuzumab, natalizumab, basiliximab, bavituximab, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, bleselumab, blinatumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, brontictuzumab, burosumab, cabiralizumab, canakinumab, cantuzumab metansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, carotuximab, cedelizumab, cergutuzumab amunaeleukin, certolizumab pegol, cetuximab, cituximab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, coltuximab ravtansine, conatumumabe, concizumab, crenezumab, coredumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab, denosumab, depatuxizumab, derlotuximab, detumomab, dinutuxidmab, diridavumab, domogrozumab, dorimomab, drozitumab, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobabcomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, emicizumab, enavatuzumab, enlimomab, enoblituzumab, enokizumab, enoticumab, ensituximab, eptitumomab, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, farlimomab, farletuzumab, fasinumab, felvizumab, fezakinumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, fresolimumab, fluranumab, futuximab, glacanezumab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab, gevokizumab, girentuximab, glembatumumab, golimumab, gomiliximab, guselkumab, ibalizumab, ibritumomab, icrucumab, idarucizumab, igovomab, imalumab, imciromab, imgatuzumab, inclacumab, indatuximab, indusatumab, inebilizumab, infliximab, intetumumab, inolimomab, inotuzumoab, ipilimumab, iratmumab, isatuximab, itolizumab, ixekizumab, keliximab, labetuzumab, lampalizumab, landelumab, landogrozumab, laprituximab, lebrikizumab, lemalesomab, lendalizumab, lenzilumab, lerdelimumab, lexatumumab, libivirumab, lifastuzumab, ligelizumab, lilotomab, lintuzumab, lirilumab, lodelcizumab, likivetmab, lorvotuzumab, lucatumumab, lulizumab, lumiliximab, lumretuzumab, mapatumumab, margetuximab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mirvetuximab, mitumomab, mogamulizumab, monalizaumab, morolimumab, motavizumab, moxetumomab, muromonab, nacolomab, namilumab, naptumomab, natatuximab, narnatumab, natlizumab, navicixizumab, navivumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, oalizumab, onartuzumab, ontuxizumab, opicinumab, opotruzumab, oregovomab, orticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibximab, palivizumab, pamrevlumab, pankomab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab, pintumomab, placulumab, plozalizumab, polatuzumab, ponezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, ranibizumab, raxibacumab, refanezumab, regavirumab, reslizumab, rilotmumab, rinucumab, risankizumab, rituximab, rivabazumab, robatumumab, roledumab, romosozumab, rontalizumab, rovalpituzumab, rovelizumab, ruplizumab, sacituzumab, samalizumab, sapelizumab, sarilumab, satumomab, secukinumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab, tadocizumab, talizumab, tamtuetmab, tanezumab, tefibazumab, telimomab, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetlomab, tezepelumab, ticilimumab, tildrakizumab, tigatuzumab, timolumab, tisotumab, tocilizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, tregalizumab, tremelimumab, trevogrumab, tocutuzumab, tuvirumab, ulituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab, vandortuzumab, vantictumab, vanucizumab, vapaliximab, varlillumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, vobarilizumab, volociximab, vorsetuzumab, votumumab, xentuzumab, zalutumumab, zanolimumab, zatuximab, ziralimumab and zolimomab. HBA's derived from combinations of the above referenced mono-specific antibodies may be used to achieve synergistic effects across various disease indications.
HBFP's incorporating the heavy chain modifications described above can be made to any target molecule. The HBFP's are particularly useful for surface-bound or circulating target proteins (e.g., on cells or viruses) in which aggregation or neutralization of the target protein induces a desired response. The desired response can be, for example, clearing of a target protein, reducing aggregation of a target protein, clearing of a cell or virus bearing a target, signal transduction through a receptor, e.g., inducing apoptosis or cytostasis, inhibiting a receptor binding to a ligand or counterreceptor, or internalization of an HBFP conjugated to a toxic agent. HBFP's can be made to the same targets as existing therapeutic fusion proteins or can be derivatized versions of therapeutic Fc fusion proteins in which one or more of the existing constant regions have been replaced by the heterologous polypeptides of the present invention.
Thus, the teachings herein can be applied to create novel HBFP's using therapeutically relevant biologic molecules having properties of interest, such as, for example, receptors, enzymes, or enzyme inhibitors (e.g. serpins) with established therapeutic efficacy. For example, the biologically active regions of R1 or R2 or both can be derived from Elocate, Alprolix, Amevive (alefacept), Enbrel® (etanercept), Orencia® (abatacept), Nulojix® (belatocept), Eylea® (aflibercept), Zaltrap® (zivaflibercept), Arcalyst® (rilonacept), IL-1 Trap, Blisbimod (a-623, AMG623), Dulaglutide (LY2189265), APG101, apocept, IL-6 trap, or C1-esterase inhibitor.
HBA's or HBFP's having the modifications described above can be produced by recombinant expression. Production of an antibody or fusion protein typically requires several expression units. For example, in the case of HBA's, one for each for the different heavy chains, and one or two for the two light chains depending whether the light chains are the same or different. The expression units can be present on separate vectors, or split among two or more vectors, or all can be present on the same vector. Production of an Fc fusion protein typically requires two expression units, one for each heavy chain. The expression units can be on the same or different vectors. For example, one heavy chain expression vector expresses one arm of the HBA or HBFP and the other heavy chain expression vector expresses the other arm of the HBA or HBFP. Typically such constructs are fused at their N-termini to a signal sequence. The modified constant regions of the HBA's or HBFP's can be introduced by methods such as site specific or cassette mutagenesis, or introduced in de novo nucleic acid synthesis. The light chain expression units typically include from N-terminus to C-terminus a signal peptide, a variable region and a light chain constant region (or the substituted heterologous polypeptide), as for standard expression of an antibody.
The order in which fusions of genetic elements is performed in building a construct encoding several components is not important. The segments can also be linked simultaneously by joining overlapping oligonucleotides encoding the respective segments in an overlapping PCR-type reaction, or by the methodology of Gibson (Gibson 2011; Merryman and Gibson 2012). In practice, once expression units encoding the heavy chain constant regions of the invention have been produced, the same expression units can be used to insert any heavy chain variable region(s) or other binding region(s) in the case of a fusion protein (and sometimes a light chain variable region) without recreating the DNA segment encoding all of the heavy chain components.
Mammalian cells are a preferred host for expressing nucleotide segments encoding HBA's or HBFP's of the invention (see Winnacker, From Genes to Clones, (VCH Publishers, N Y, 1987)). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2/0 and NS0. Preferably, the cells are nonhuman. Preferably, an HBA or HBFP of the invention is expressed from a monoclonal cell line.
Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).
Cells are transfected with one or more vectors encoding the HBA or HBFP to be expressed. For a multi-chain antibody, the heavy and light chains can be expressed on the same or separate vectors. For expression of multi-subunit complexes, the DNA encoding the components of the complexes (i.e., different antibodies or fusion proteins) can be on the same or different vectors.
HBA's or HBFP's can be made by culturing host cells, such as the cells discussed above, under conditions so as to express the nucleic acid(s) encoding the HBA or HBFP, and recovering the antibody or fusion protein. In some methods, one or more HBA or HBFP chains are separately isolated and assembled, outside of the cell, for example, in the supernatant or in vitro. In some methods, HBA or HBFP chains are expressed, processed to remove signal peptides, assembled and secreted from host cells. It is believed that association of different heavy chains, association between heavy and light chains occur at least predominantly within cells so that HBA's are secreted in a fully associated state.
HBA's or HBFP's can be purified from cell culture supernatants by conventional antibody purification methods. The purification can include a chromatography step using protein A or protein G as the affinity reagent, provided that the requisite protein A or protein G binding sites are retained in the heterologous heavy chain. In addition, affinity chromatography directed towards the X/Y, or p/q, or m/n, or any combination of the above binding pairs, could be employed sequential to, or instead of protein-A or protein-G chromatography, to effect product purity during manufacture of HBA or HBFP described supra. Conventional antibody or protein purification procedures, such as ion exchange, hydroxyapatite chromatograph or HPLC can also be used (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).
The HBA's or HBFP's can include conjugated therapeutic moieties that can be used to treat, combat, ameliorate, prevent or improve an unwanted condition or disease in a patient. For example, HBA's or HBFP's can be conjugated to a toxic agent. Toxic agents can be cytotoxic or cystostatic. Some example of toxic agents include antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, camptothecins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, pre-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. Conjugates of an antibody and toxic agent can be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). A toxic agent can also be linked to an antibody via a linker, which may be cleavable under intracellular conditions (US 2003-0083263, 2005-0238649 and 2005-0009751). Many of the above toxic agents are only effective or most effective when internalized within a cell. The HBA's of the invention can be internalized by binding to cellular receptors, for example, crosslinking of cellular receptors can promote internalization. The HBA's can also be coupled to one or more other antibodies or to a detectable label.
The HBA's or HBFP's disclosed herein can be used for treating or effecting prophylaxis of a disease in a patient having or at risk for the disease targeted by any of the mono-specific antibodies or fusion proteins disclosed herein. For example, the HBA's or HBFP's can be used to treat or effect prophylaxis of a patient having or at risk for having cancer, an autoimmune or inflammatory condition, a neurodegenerative disease, an infectious disease, osteoporosis, dyslipidemia, macular degeneration, a blood coagulation disorder, a cardiovascular disease or disorder, an organ transplant, diabetes, influenza, a muscle wasting disorder or a gastrointestinal disease or disorder by administering the HBA or HBFP in a therapeutically effective regime.
The HBA's or HBFP's of the invention can be used for treating cancers including those for which commercial antibodies or fusion proteins mentioned above have been used. The methods can be used to treat one or more hematological malignancies, such as leukemia (e.g., T cell large granular lymphocyte leukemia), lymphoma (Hodgkin's or Non-Hodgkin's), or multiple myeloma. Some HBA's or HBFP's are suitable for treating solid tumors such as, for example, skin cancer (e.g., melanoma), ovarian, endometrial, bladder, breast, rectum, colon, gastric, pancreatic, lung, thymus, kidney and brain. Some HBA's or HBFP's can be used to treat prostate cancer, breast cancer, colorectal cancer, non-small cell lung carcinoma, gastrointestinal cancer, metastatic cancer, squamous cell carcinoma, head and neck cancer, solid tumors, glioblastoma, neuroblastoma, testicular cancer, adrenocortical carcinoma or pancreatic cancer. Other hematological cancers suitable for treatment with some HBA's of the invention include acute myelogenous leukemia and chronic lymphocytic leukemia.
The HBA's or HBFP's of the invention can also be used for suppressing various undesirable immune responses including those in which the therapeutic antibodies mentioned above have been used.
One category of immune disorders treatable by some HBA's or HBFP's of the invention is transplant rejection. When allogeneic cells or organs (e.g., skin, kidney, liver, heart, lung, pancreas and bone marrow) are transplanted into a recipient (i.e., the donor and recipient are different individuals from the same species), the recipient's host immune system is likely to mount an immune response to foreign antigens in the transplant (host-versus-graft disease) leading to destruction of the transplanted tissue. The HBA's or HBFP's of the present invention are useful, inter alia, to block alloantigen-induced immune responses in the recipient.
A related use for HBA's or HBFP's of the present invention is in modulating the immune response involved in “graft versus host” disease (GVHD). GVHD is a potentially fatal disease that occurs when immunologically competent cells are transferred to an allogeneic recipient. In this situation, the donor's immunocompetent cells may attack tissues in the recipient. Tissues of the skin, gut epithelia and liver are frequent targets and may be destroyed during the course of GVHD. The disease presents an especially severe problem when immune tissue is being transplanted, such as in bone marrow transplantation; but less severe GVHD has also been reported in other cases as well, including heart and liver transplants.
A further situation in which immune suppression is desirable is in treatment of autoimmune or inflammatory diseases such as Crohn's disease, ulcerative colitis, inflammatory bowel disease, multiple sclerosis, stiff man syndrome, rheumatoid arthritis, myasthenia gravis, Type I diabetes, Graves' disease, myasthenia gravis, Sjogren's syndrome and lupus erythematosus. In these diseases, the body develops a cellular and/or humoral immune response against one of its own antigens leading to destruction of that antigen, and potentially crippling and/or fatal consequences. Autoimmune diseases are treated by administering the applicable HBA's or HBFP's of the invention.
Other immune disorders treatable by the HBA's or HBFP's of the invention include asthma, allergies, celiac disease, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis and uveitis. Celiac disease, psoriasis and uveitis are autoimmune diseases.
Some HBA's or HBFP's are useful for the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, corticobasal degeneration, frontotemporal lobar degeneration, Lewy body disease, Pick's disease, progressive supranuclear palsy, multiple system atrophy and ALS.
The HBA's or HBFP's can also be used for treatment of pathogenic infections, such as viral, bacterial, protozoan or fungal infection. Some example of viral infections include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein Barr virus), adenovirus, XMRV, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, MLV-related Virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus. Some examples of bacterial infections include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, Lymes disease bacteria, streptococci, or neisseria. Some examples of pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys. Examples of protozoa include Cryptosporidium, Giardia lamblia and plasmodium.
The specific disease targeted will depend on the individual properties of the antibody selected for conversion into an HBA. Pharmaceutical compositions comprising the HBA's or HBFP's of the invention would be administered to the patient in a regime (dose, frequency and route of administration) effective to reduce the risk, lessen the severity or delay the onset and/or progression of at least one sign or symptom of the disease.
HBA's or HBFP's are administered in an effective regime, meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of a disorder. If a patient is already suffering from a disorder, the regime can be referred to as a therapeutically effective regime. If the patient is at elevated risk of the disorder relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients.
Exemplary dosages for an HBA or HBFP are 0.01-20, or 0.5-5, or 0.01-1, or 0.01-0.5 or 0.05-0.5 mg/kg body weight (e.g., 0.1, 0.5, 1, 2, 3, 4 or 5 mg/kg) or 10-1500 mg as a fixed dosage. The dosage depends on the condition of the patient and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic and whether the disorder is acute or chronic, among other factors.
Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Administration into the systemic circulation by intravenous or subcutaneous administration is preferred. Intravenous administration can be, for example, by infusion over a period such as 30-90 min.
The frequency of administration depends on the half-life of the HBA or HBFP in the circulation, the duration of effect, the condition of the patient and the route of administration among other factors. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient's condition or progression of the disorder being treated. An exemplary frequency for intravenous administration is between weekly and quarterly over a continuous cause of treatment, although more or less frequent dosing is also possible. For subcutaneous administration, an exemplary dosing frequency is daily to monthly, although more or less frequent dosing is also possible.
The number of dosages administered depends on whether the disorder is acute or chronic and the response of the disorder to the treatment. For acute disorders or acute exacerbations of chronic disorders between 1 and 10 doses are often sufficient. Sometimes a single bolus dose, optionally in divided form, is sufficient for an acute disorder or acute exacerbation of a chronic disorder. Treatment can be repeated for recurrence of an acute disorder or acute exacerbation. For chronic disorders, an antibody can be administered at regular intervals, e.g., weekly, fortnightly, monthly, quarterly, every six months for at least 1, 5 or 10 years, or the life of the patient.
Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection). The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The HBA's can be used for detecting their target molecule in the context of clinical diagnosis or treatment or in research. For example, the HBA's can be used to detect a cancer-related antigen as an indication a patient is suffering from an immune mediated disorder amenable to treatment. The HBA's can also be sold as research reagents for laboratory research in detecting targets and their response to various stimuli. In such uses, HBA's can be labeled with fluorescent molecules, spin-labeled molecules, enzymes or radioisotypes, and can be provided in the form of kit with all the necessary reagents to perform the assay. The HBA's can also be used to purify their target antigens e.g., by affinity chromatography.
All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.
The VH and VL domains from adalimumab1 are selected as the antigen specificity determinants for a construct, as it is a well characterized antibody of known crystal structure (pdb id 3WD5). A discrepancy in sequence of adalimumab VH at residue 82 is noted between the pdb entry and the drugbank.ca web site, with pdb indicating D82, and the drugbank entry indicating Q82. Structural inspection of 3WD5 revealed residue 82 is framework 3, and likely to be of minimal consequence for antigen recognition, Q82 was retained for testing in the prototype construct. Sequence analysis of adalimumab V regions revealed VH is closest to human sub-group 3, and VL is closest to human kappa chains sub-group 1 (Kabat et al. 1991). Leader peptides for adalimumab V-regions were selected from a human anti-HIV-1 gp41 antibody designated 3D6 as its V region sequences belonged to the same subgroup as adalimumab (Felgenhauer et al. 1990; Kabat et al. 1991). The amino-acid sequences of L and V regions of adalimumab are reverse translated and codon optimized for human codon usage2, and fused to human Cκ and Cγ1 encoding sequence. The full sequences of Adalimumab light chain and heavy chain protein and DNA sequences are provided in
In order to effect heterodimer formation between two heavy chains, the CH3 domains are replaced with MHC-I α3 domain or β2 microglobulin domain, taking into consideration structural information regarding flexible loops separating distinct domains. The DNA and predicted protein sequences for these MHC-I α3 domain from HLA-A (A*68 allele) or β2 microglobulin hybrid adalimumab heavy chains are provided in
The CH3 domains are replaced with MHC-II α2 domain or MHC II β2 domain, taking into consideration structural information regarding flexible loops separating distinct domains. The DNA and predicted protein sequences for these MHC-II α2 domain (derived from HLA-DR alpha) or MHC II β2 domain (derived from HLA-DR beta1) hybrid adalimumab heavy chains are provided in
The CH3 domains are replaced with T-cell antigen receptor (TCR) Ca domain or TCR Cβ domain, taking into consideration structural information regarding flexible loops separating distinct domains. The DNA and predicted protein sequences for these TCR Cα domain or TCR Cβ domain hybrid adalimumab heavy chains are provided in
The CH3 domains are replaced with T-cell antigen receptor (TCR) Cy domain or TCR Cδ domain, taking into consideration structural information regarding flexible loops separating distinct domains. The DNA and predicted protein sequences for these TCR Cγ domain or TCR Cδ domain hybrid adalimumab heavy chains are provided in
The CD3 ε-chain ectodomain pairs with CD3 γ-chain as well as with CD3 δ-chain ectodomain (Arnett et al. 2004; Kjer-Nielsen et al. 2004). The CD3 γ, δ, and ε-chain ectodomains are Ig SF members and their Ig fold is of pfam id I-set (Table 1), in contrast with CH3 (pfam id C-set). The potential for promoting heterodimer formation in Ig heavy chains as CH3 domain swaps with CD3 γ, δ, & ε-chain ectodomains is tested. The CH3 domains are replaced with CD3 ε, γ, or δ-ectodomain, taking into consideration structural information regarding flexible loops separating distinct domains. The DNA and predicted protein sequences for these CD3 ε-ectodomain, γ-ectodomain, or δ-ectodomain hybrid adalimumab heavy chains are provided in
Recombinant synthetic DNAs were purchased from Blue Heron as clones in pUC19. These clones provided the cassettes encoding the domains listed in examples 1-6 for making bi-functional antibody constructs. The clones are listed in table 13. All synthetic clones and constructs derived from the clones were sequence verified prior to their use in subsequent steps throughout the course of this work.
Forward and reverse per primers used for assembling and cloning the bi-functional antibody expression constructs into target mammalian expression vector pcDNA3.1-are listed in table 14.
The strategy for assembling the full-length light and heavy chains of the positive control antibody (adalimumab) listed in example 1 is illustrated in
The strategy for amplification of paired heavy chains cassettes listed in examples 2-6, using templates and primers listed in tables 2 and 3 respectively, is illustrated in
The strategy for assembling paired heterodimeric heavy chains listed in examples 2-6 from amplified cassette fragments (
The amplified fragments were purified from the gels shown in
Conditioned media was harvested from cells 72 h post transfection for detection of expressed and secreted proteins by immunoassay, and transfected cells were harvested for analysis of GFP reporter by flow cytometry to check transfection efficiency across all reactions listed in Table 16. The GFP expression in transfected cells is shown in
Expression of recombinant protein by the transfected cells, and concentration of protein in conditioned media was confirmed by ELISA. ELISA for protein concentration was carried out in 96 well plates coated overnight with 100 uL of 10 ug/mL Goat anti human IgG (Jackson Immune lab, Cat. 109-005-008) per well (1 ug/well). The coated plates were blocked, washed, incubated with 100 ul/well conditioned media in triplicate from transfected cells, or human IgG as concentration standard for 1 hr at room temperature, washed again and incubated with 100 uL of 1:10000 HRP conjugated second antibody (Jackson immune lab, Cat. 109-035-003) in blocking buffer at RT for 1 hr as detection reagent, washed and developed by adding 100 uL of H2O2-Amplx Red HRP substrate for 30 min at RT in the dark. Human IgG was used as concentration standard in the ELISA at a range of concentrations. Results from concentration assessment of conditioned media from transfections listed in Table 16 are presented in
Biochemical characterization of the secreted heavy chains homo and hetero-dimers was carried out by immunoprecipitation of conditioned media, followed by SDS-PAGE. Immunoprecipitation was carried out on an equal amount of protein (˜10 Mg/sample, based on concentration as determined in
Functional binding of the recombinant antibodies as either heavy chain homodimers, or heavy chain heterodimers was confirmed by antigen recognition in an ELISA format. The antigen, TNFα was immobilized on plates followed by incubation with equal concentrations of conditioned media from cells co-transfected with adalimumab light chain plus either hybrid heavy chain monomers (homodimers), or paired hybrid heavy chains (heterodimers), as indicated in Table 16. For confirming binding of samples to immobilized antigen, all samples were diluted to a nominal starting 1 μg/ml, and 2× serial dilutions were applied to the antigen coated plates. In parallel, the samples were also assayed for actual concentration in the assay by capture on Goat anti-Human antibody coated plates, and the concentration was determined relative to a control human antibody of known concentration as a reference standard. The results, shown in
The present application is a US national stage entry of PCT/US2018/038833 filed Jun. 21, 2018, which claims the benefit of U.S. 62/523,220 filed Jun. 21, 2017, which is incorporated by reference in its entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/38833 | 6/21/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62523220 | Jun 2017 | US |
