Patent Application
20200317792
This invention relates to antibodies, methods of predicting the sequences of variant antibodies, and methods of making same. The invention relates more particularly to variant antibodies to human epidermal growth factor receptor (EGFR).
Antibodies have emerged as a leading class of biotherapeutics (Aggarwal, 2014), but despite a long history and large market presence, developers of new antibodies continue to face a number of challenges (Lu et al., 2012). One such issue is immunogenicity risk, wherein anti-biotherapeutic antibody responses can reduce therapeutic efficacy and manifest a range of other detrimental side effects (Jawa et al., 2013, Pendley et al., 2003, Roskos et al., 2004, Schellekens, 2002, Swann et al., 2008). This issue has been in part, though not entirely (Harding et al., 2010, Hwang and Foote, 2005), addressed by the establishment of well-defined humanization methods (Baca et al., 1997, Dall'Acqua et al., 2005, Dennis, 2010, Gonzales et al., 2004, Jones et al., 1986, Khee Hwang et al., 2005, Lazar et al., 2007, Osbourn et al., 2005, Roguska et al., 1994) and the advent of “fully human” antibodies (Duvall et al., 2011, Feldhaus et al., 2003, Lee et al., 2014, Li et al., 2006, Lonberg, 2008, McCafferty et al., 1990). Stability is another determinant of therapeutic antibody “developability” (Jarasch et al., 2015), as loss of structural integrity can degrade binding activity, cause aggregation, and generally undermine therapeutic potential (Frokjaer and Otzen, 2005, Hermeling et al., 2004). Biotherapeutic aggregation and immunogenicity are often correlated, as protein aggregates, resulting from poor thermostability or other factors, can exacerbate classical anti-drug antibody responses and produce detrimental responses via non-classical pathways (Ratanji et al., 2013, Rosenberg, 2006).
Despite accelerating growth in fully human antibody technologies, immunization of animal models followed by antibody cloning and humanization is simple, convenient, and widely used (Nelson et al., 2010). Humanization via complementarity determining region (CDR) grafting is a common strategy. Unfortunately, CDR grafting often leads to considerable decreases in thermostability and binding affinity, requiring back substitution of non-human residues to mitigate loss of functionality (Clark, 2000). Thus successful engineering of humanized antibodies requires simultaneous consideration of multiple objectives: humanness, thermostabililty, and binding affinity.
The invention is based in part on the development of variant cetuximab sequences. Variants within the invention show enhanced thermostability (up to a 6° C. higher melting point) and substantially improved humanness, i.e., they resemble more closely the germline form of the heavy chain and/or light chain gene than a starting, or parent, cetuximab antibody. In addition, binding affinities are near to or even better than that of the parental antibody (up to 1.5 times superior KD).
In one aspect, the invention provides an isolated variant cetuximab antibody monoclonal antibody or antigen binding portion thereof. The antibody or antigen binding fragment includes a heavy chain and/or a light chain. The heavy chain and/or light chain is more similar in sequence to a germ-line EGFR antibody sequence than is a counterpart cetuximamb polypeptide, e.g., a polypeptide comprising SEQ ID NO:1 or SEQ ID NO:2.
In another aspect, the invention provides an isolated monoclonal antibody or antigen binding portion thereof. The antibody or antigen-binding portion thereof, the antibody or antigen binding fragment comprising a heavy chain and a light chain that is more thermostable than the corresponding isolated monoclonal antibody or antigen binding portion thereof comprising SEQ ID NO:1 and SEQ ID NO:2.
In a further aspect, the invention provides an isolated monoclonal antibody or antigen binding portion thereof that heavy chain and/or light chain is both more similar in sequence to a germ-line EGFR antibody sequence than is a counterpart polypeptide comprising SEQ ID NO:1 or SEQ ID NO:2 and is also more thermostable than the corresponding isolated monoclonal antibody or antigen binding portion thereof comprising SEQ ID NO:1 and SEQ ID NO:2.
In some embodiments, the heavy chain or light chain is more similar in sequence to a germ-line EGFR antibody sequences, e.g., a germline antibody sequence selected from the group consisting of SEQ ID NOs.:19-22 and 23, than is SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the heavy or light chain sequence comprises the sequences disclosed in Table 1.
In some embodiments, the isolated monoclonal antibody further comprises a heavy chain CDR1 (HCDR1) region selected from the group consisting of SEQ ID NO:13, SEQ ID NO: 14 and SEQ ID NO:15.
In some embodiments, the isolated monoclonal antibody further comprises a light chain CDR1 (LCDR1) region selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 17 and SEQ ID NO:18.
In a still further aspect, the invention provides an isolated nucleic acid sequence encoding a variant cetuximab monoclonal antibody. The nucleic acid sequence may encode a heavy chain antibody, a light chain antibody, or both.
Also provided by the invention is a recombinant expression vector that includes an isolated nucleic acid sequence encoding a variant cetuximab monoclonal antibody.
In another aspect the invention includes a recombinant expression host cell that includes a nucleic acid sequence encoding a variant cetuximab monoclonal antibody, e.g., a recombinant expression vector with a variant cetuximab monoclonal antibody encoding sequence. A host cell may include nucleic acid sequences encoding heavy chain variant cetuximab polypeptides, nucleic acid sequences encoding light chain variant cetuximab polypeptides, or both heavy chain and light chain variant cetuximab polypeptides.
In a further aspect, the invention provides a pharmaceutical composition comprising a variant cetuximab antibody, or antigen-binding portion thereof, and a pharmaceutically acceptable carrier.
In a still further aspect, the invention provides a method of treating an EGFR-associated disease or disorder. The method includes administering an effective amount of a pharmaceutical composition comprising a variant cetuximab antibody, or antigen-binding portion thereof, to a patient in need thereof.
In some embodiments, the EGFR-associated disease or disorder is a cancer or tumor expressing EGFR. The cancer or tumor can be, e.g., glioblastoma, ductal or intraductal breast carcinoma, non-small squamous cell carcinoma, ovarian carcinoma, prostate cancer, or squamous cell carcinoma of the head and neck.
In some embodiments, the disease or disorder is an inflammatory or autoimmune disease or disorder, e.g., chronic obstructive pulmonary disease, systemic lupus erythematosus, rheumatoid arthritis, splanchnic artery occlusion shock, spinal cord injury, type 1 diabetes, or multiple sclerosis.
In some embodiments, t the pharmaceutical composition is administered in combination with one or more additional therapeutic agents. The additional therapeutic agent can be, e.g., IL-18 antagonist, IL-12 antagonist, TNF antagonist, methotrexate, corticosteroid, cyclosporin, rapamycin, FK506, and/or non-steroidal anti-inflammatory agents.
The additional agent or agents can be administered either concurrently with the variant cetuximab antibody pharmaceutical composition or at a separate time.
Also provided by the invention is a method making a variant cetuximab antibody. The method includes culturing a cell under conditions that allow for expression and function of a variant cetuximab antibody. In some embodiments, the method includes purifying the expressed variant cetuximab antibody.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
We applied CoDAH to engineer variants of cetuximab, a chimeric antibody targeting the epidermal growth factor receptor (EGFR) (Kirkpatrick et al., 2004). The computationally designed variants consistently exhibit enhanced thermostabilities (up to 6° C.) and substantially improved humanness, along with binding affinities near to or even better than that of the parental antibody (up to 1.5 times better KD).
The term “antibody” is used herein in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies) of any isotype such as IgG, IgM, IgA, IgD and IgE, polyclonal antibodies, multispecific antibodies, chimeric antibodies, and antibody fragments. An antibody reactive with a specific antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the antigen or an antigen-encoding nucleic acid.
A typical IgG antibody is comprised of two identical heavy chains and two identical light chains that are joined by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three segments called “complementarity-determining regions” (“CDRs”) or “hypervariable regions”, which are primarily responsible for binding an epitope of an antigen. They are usually referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The more highly conserved portions of the variable regions are called the “framework regions”.
As used herein, “VH” or “VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, dsFv, Fab, Fab′ or F(ab′)2 fragment. Reference to “VL” or “VL” refers to the variable region of the immunoglobulin light chain of an antibody, including the light chain of an Fv, scFv, dsFv, Fab, Fab′ or F(ab′)2 fragment.
A “polyclonal antibody” is an antibody which was produced among or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes producing non-identical antibodies. Usually, polyclonal antibodies are obtained directly from an immunized animal.
A “monoclonal antibody”, as used herein, is an antibody obtained from a population of substantially homogeneous antibodies, i.e. the antibodies forming this population are essentially identical except for possible naturally occurring mutations which might be present in minor amounts. These antibodies are directed against a single epitope and are therefore highly specific.
An “epitope” is the site on the antigen to which an antibody binds. If the antigen is a polymer, such as a protein or polysaccharide, the epitope can be formed by contiguous residues or by non-contiguous residues brought into close proximity by the folding of an antigenic polymer. In proteins, epitopes formed by contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by non-contiguous amino acids are typically lost under said exposure.
The “humanness” of an antibody is the degree to which it retains the sequence of a germ-line heavy chain or light chain counterpart antibody.
The term “humanized antibody”, as used herein, refers to a chimeric antibody which contains minimal sequence derived from non-human immunoglobulin. The goal of humanization is a reduction in the immunogenicity of a xenogeneic antibody, such as a murine antibody, for introduction into a human, while maintaining the full antigen binding affinity and specificity of the antibody. Humanized antibodies, or antibodies adapted for non-rejection by other mammals, may be produced using several technologies such as resurfacing and CDR grafting. As used herein, the resurfacing technology uses a combination of molecular modeling, statistical analysis and mutagenesis to alter the non-CDR surfaces of antibody variable regions to resemble the surfaces of known antibodies of the target host. The CDR grafting technology typically involves substituting the complementarity determining regions of, for example, a mouse antibody, into a human framework domain, e.g., see WO 92/22653. Humanized chimeric antibodies preferably have constant regions and variable regions other than the complementarity determining regions (CDRS) derived substantially or exclusively from the corresponding human antibody regions and CDRs derived substantially or exclusively from a mammal other than a human.
Strategies and methods for the resurfacing of antibodies, and other methods for reducing immunogenicity of antibodies within a different host, are disclosed in U.S. Pat. No. 5,639,641, which is hereby incorporated in its entirety by reference. Briefly, in a preferred method, (1) position alignments of a pool of antibody heavy and light chain variable regions is generated to give a set of heavy and light chain variable region framework surface exposed positions wherein the alignment positions for all variable regions are at least about 98% identical; (2) a set of heavy and light chain variable region framework surface exposed amino acid residues is defined for a rodent antibody (or fragment thereof); (3) a set of heavy and light chain variable region framework surface exposed amino acid residues that is most closely identical to the set of rodent surface exposed amino acid residues is identified; (4) the set of heavy and light chain variable region framework surface exposed amino acid residues defined in step (2) is substituted with the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3), except for those amino acid residues that are within 5.ANG. of any atom of any residue of the complementarity-determining regions of the rodent antibody; and (5) the humanized rodent antibody having binding specificity is produced.
Antibodies can be humanized using a variety of other techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E. A., 1991, Molecular Immunology 28(415): 489-498; Studnicka G. M. et al., 1994, Protein Engineering, 7(6): 805-814; Roguska M. A. et al., 1994, PNAS, 91: 969-973), and chain shuffling (U.S. Pat. No. 5,565,332). Human antibodies can be made by a variety of methods known in the art including phage display methods. See also U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and international patent application publication numbers WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (said references incorporated by reference in their entireties).
Variant cetiuximab molecules according to the invention are shown below. Heavy chain cetuximab variants correspond to SEQ ID Nos: 3-7. Light chain cetuximab variants correspond to SEQ ID Nos: 8-12. Previously described cetuximab heavy chain and light sequences are shown as SEQ ID Nos. 1 and 2, respectively. As is shown below, the heavy and light chain variants are interchangeable.
Nucleic acids encoding humanized and thermostabilized antibodies of the invention are provided. In one embodiment, the nucleic acid molecule encodes a heavy and/or a light chain of a humanized and thermostabilized immunoglobulin. In a preferred embodiment, a single nucleic acid encodes a heavy chain of a humanized and thermostabilized immunoglobulin and another nucleic acid molecule encodes the light chain of a humanized and thermostabilized immunoglobulin.
In order to express the heavy and/or light chain of the humanized and thermostabilized antibodies of the invention, the polynucleotides encoding said heavy and/or light chains are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational sequences. Expression vectors include plasmids, YACs, cosmids, retrovirus, EBV-derived episomes, and all the other vectors that the skilled man will know to be convenient for ensuring the expression of said heavy and/or light chains. The skilled man will realize that the polynucleotides encoding the heavy and the light chains can be cloned into different vectors or in the same vector. In a preferred embodiment, said polynucleotides are cloned in the same vector.
Polynucleotides of the invention and vectors comprising these molecules can be used for the transformation of a suitable mammalian host cell. Transformation can be by any known method for introducing polynucleotides into a cell host. Such methods are well known of the man skilled in the art and include dextran-mediated transformation, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, biolistic injection and direct microinjection of DNA into nuclei.
The antibodies of the present invention include both the full length antibodies discussed above, as well as epitope-binding fragments thereof. As used herein, “antibody fragments” include any portion of an antibody that retains the ability to bind to the epitope recognized by the full length antibody, generally termed “epitope-binding fragments.” Examples of antibody fragments include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising either a VL or VH region. Epitope-binding fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains.
Such fragments may contain one or both Fab fragments or the F(ab′)2 fragment. Preferably, the antibody fragments contain all six CDRs of the whole antibody, although fragments containing fewer than all of such regions, such as three, four or five CDRs, are also functional. Further, the fragments may be or may combine members of any one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof.
Fab and F(ab′)2 fragments may be produced by proteolytic cleavage, using enzymes such as papain (Fab fragments) or pepsin (F(ab′)2 fragments).
The “single-chain FVs” (“scFvs”) fragments are epitope-binding fragments that contain at least one fragment of an antibody heavy chain variable region (VH) linked to at least one fragment of an antibody light chain variable region (VL). The linker may be a short, flexible peptide selected to assure that the proper three-dimensional folding of the VL and VH regions occurs once they are linked so as to maintain the target molecule binding-specificity of the whole antibody from which the single-chain antibody fragment is derived. The carboxyl terminus of the VL or VH sequence may be covalently linked by a linker to the amino acid terminus of a complementary VL or VH sequence.
Single-chain antibody fragments of the present invention contain amino acid sequences having at least one of the variable or complementarity determining regions (CDRs) of the whole antibodies described in this specification, but lack some or all of the constant domains of those antibodies. These constant domains are not necessary for antigen binding, but constitute a major portion of the structure of whole antibodies. Single-chain antibody fragments may therefore overcome some of the problems associated with the use of antibodies containing a part or all of a constant domain. For example, single-chain antibody fragments tend to be free of undesired interactions between biological molecules and the heavy-chain constant region, or other unwanted biological activity. Additionally, single-chain antibody fragments are considerably smaller than whole antibodies and may therefore have greater capillary permeability than whole antibodies, allowing single-chain antibody fragments to localize and bind to target antigen-binding sites more efficiently. Also, antibody fragments can be produced on a relatively large scale in prokaryotic cells, thus facilitating their production. Furthermore, the relatively small size of single-chain antibody fragments makes them less likely to provoke an immune response in a recipient than whole antibodies.
Single-chain antibody fragments may be generated by molecular cloning, antibody phage display library or similar techniques well known to the skilled artisan. These proteins may be produced, for example, in eukaryotic cells or prokaryotic cells, including bacteria. The epitope-binding fragments of the present invention 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 particular, such phage can be utilized to display epitope-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an epitope-binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled 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 epitope-binding fragments of the present invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods, 182: 41-50; Ames et al., 1995, J. Immunol. Methods, 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol., 24: 952-958; Persic et al., 1997, Gene, 187: 9-18; Burton et al., 1994, Advances in Immunology, 57: 191-280; WO/1992/001047; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/118619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
After phage selection, the regions of the phage encoding the fragments can be isolated and used to generate the epitope-binding fragments through expression in a chosen host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, using recombinant DNA technology, 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 WO 92/22324; Mullinax et al., 1992, BioTechniques, 12(6): 864-869; Sawai et al., 1995, AJRI, 34: 26-34; and Better et al., 1988, Science, 240:1041-1043; said references incorporated by reference in their entireties. 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 in Enzymology, 203: 46-88; Shu et al., 1993, PNAS, 90: 7995-7999; Skerra et al., 1988, Science, 240:1038-1040.
Cetuximab, also known as Erbitux® and IMC-C225 or CMAB009, is a chimeric human/mouse antibody that binds to epidermal growth factor receptor (EGFR). Examples disclosing cetuximab and its use are disclosed in WO 96/040210; WO 99/060023; WO 00/069459; WO 02/045653; WO 04/085474; WO 05/051355; WO 07/127936; WO 07/147001; WO 09/062083; WO 10/056893; and WO 10/080463, each of which is incorporated herein by reference in its entirety.
As used herein, “thermostabilized” or “thermostability” refers to the quality of a protein or antibody to resist chemical or physical change as a result of increasing temperature. For the purposes of this invention, alterations to the amino acid sequence of an antibody may be made to increase the thermostability of said antibody compared to the parent antibody. Thermostability may be determined by any known method in the field, including the measurement of the antibody melting temperature (TM). Improvements in thermostability include increases in the TM by greater than or equal to 0.1° C. to greater than or equal to 10.0° C.
As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cells, inhibiting metastasis of neoplastic cells, shrinking or decreasing the size of a tumor, remission of cancer, decreasing symptoms resulting from cancer, increasing the quality of life of those suffering from cancer, decreasing the dose of other medications required to treat cancer, delaying the progression of cancer, curing a cancer, and/or prolong survival of patients having cancer.
As used herein, an “effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results. In more specific aspects, an effective amount prevents, alleviates or ameliorates symptoms of disease, and/or prolongs the survival of the subject being treated. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms of a disease such as, for example, cancer including, for example without limitation, gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, squamous cell head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colorectal cancer, oral cancer, skin cancer, and melanoma, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and/or delaying the progression of the cancer in patients. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective dosage” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
An “individual” or a “subject” is a mammal, more preferably, a human. Mammals also include, but are not limited to, farm animals (e.g., cows, pigs, horses, chickens, etc.), sport animals, pets, primates, horses, dogs, cats, mice and rats.
The invention will be further illustrated in the following non-limiting examples.
CoDAH (Computationally-Driven Antibody Humanization) (Choi, Hua, Sentman, Ackerman and Bailey-Kellogg, 2015, incorporated herein by reference) is a structure-based protein design method that optimizes variants of a parental Fv by selecting sets of allowed mutations according to in silico evaluation of their effects on humanness and stability.
The humanness of an antibody is assessed in terms of its human string content score (Lazar G A, Desjarlais J R, Jacinto J, Karki S, Hammond P W. A molecular immunology approach to antibody humanization and functional optimization. Mol Immunol 2007; 44:1986-98) (
The indicator function l{ } is 1 if the predicate is true, i.e., the amino acid in the target antibody is the same as the corresponding one in the germline, and 0 otherwise. So the maximum score contributed by a peptide is 9, if all 9 of its amino acids are identical to corresponding amino acids in some germline sequence.
The HSC score of an entire antibody chain v is then the average over all its constituent peptides, scaled to a percentage (0-100).
Antibody stability (
The CDRs are not allowed to mutate during humanization. To identify CDRs, antibody sequences are annotated by the Kabat numbering scheme using the AbNum program. (Abhinandan K, Martin A C. Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol 2008; 45:3832-9). Light chain CDRs are defined as residues 24-34 (L1), 50-56 (L2) and 89-97 (L3), and heavy chain CDRs are composed of residues 31-35 (H1), 50-65 (H2) and 95-102 (H3).
At each position, any amino acid that appears in some human germline antibody sequence at the corresponding aligned position is considered as a possible substitution. It is also possible to filter the set based on those that are sufficiently frequent, appear in particular germlines, or based on other criteria as desired.
The humanization algorithm identifies the Pareto optimal protein designs, (He L, Friedman A M, Bailey-Kellogg C. A divide-and-conquer approach to determine the Pareto frontier for optimization of protein engineering experiments. Proteins 2012; 80:790-806) i.e., those making the best trade-offs between the 2 competing objectives of HSC score and rotamer-based energy. The algorithm follows a “sweep” approach analogous to that previously developed for enzyme deimmunization. (Parker A S, Choi Y, Griswold K E, Bailey-Kellogg C. Structure-guided deimmunization of therapeutic proteins. J Comput Biol 2013; 20:152-65). Initially, the HSC score (Eq. 1, above) is calculated for the original target antibody sequence. The lowest-energy variant with a better HSC score than that of the original is then optimized, moving one step to the right on the curve in
In order to optimize the minimum-energy variant achieving at least a specified HSC score, the integer programming formulation of Parker et al. (supra) is adapted to use HSC. The integer program represents a variant as a set of rotamers, some from the original sequence and some as substitutions, encoded by a set of binary variables: Si,r indicates whether or not the variant uses rotamer r at position i. In order to incorporate pairwise energy terms, the integer program also includes pair variables: pir,jt indicates whether or not the variant uses both rotamer r at i and t at j. Finally, in order to assess and thereby constraint HSC score, the “window” binary variable Wi,p is defined to indicate whether or not amino acids spanning positions i to i+l−1 (l=9 in this case) correspond to linear peptide p (i.e., a sequence of 9 amino acids). Only peptides with HSC scores better than the original sequence are allowed.
The objective function for the integer program is to minimize the energy E, computed as:
where Ei(r) is the energy of rotamer r at position i and Eij(r,t) the pairwise energy between rotamers r at i and t at j.
During the sweep, the next variant is constrained to improve the HSC score beyond the value H for the previous variant:
The variables are further constrained as follows, in order to ensure that the pair and window variables are consistent with the singleton variables, and that only one rotamer is taken at each position:
where p[h] is the amino acid type at position h in peptide p, and a(r) is that of rotamer r.
Additionally the mutation load m can be constrained, specifying the number of rotamers that are not of the corresponding original amino acid type:
where t[i] is the amino acid at position i in the original target.
The Human String Content (HSC) score (Lazar, Desjarlais, Jacinto, Karki and Hammond, 2007) assesses humanness as the extent of identity of nonamer peptides within a variant to corresponding nonamers within human germline antibody sequences. The heavy (VH) and light chain (Vκ) variable regions of cetuximab were aligned to a total of 212 unique VH and 85 Vκ human germline antibody sequences extracted from publicly available databases including VBASE and IMGT (Poiron et al., 2010, Retter et al., 2005).
Structure-based rotameric energies assess the energetic impacts of mutations according to a molecular mechanics force field. CoDAH uses one- and two-body energy potentials defined by the AMBER force field (Pearlman et al., 1995, Qiu et al., 1997) as implemented by OSPREY (Chen et al., 2009, Gainza et al., 2012). The Fv portion from a crystal structure of cetuximab (PDB code: 1YY8) was used to parameterize these energies.
CoDAH generated a set of 43 Pareto optimal (He et al., 2012, Parker et al., 2013, Parker et al., 2010) integrated VH/Vκ cetuximab variants, those making the best trade-offs between the HSC score and rotameric energy (
As a comparator, a panel of humanized variants was also constructed using a conventional approach to CDR grafting. The CDRs of cetuximab (defined by the Kabat numbering scheme, see Table 2 below) were grafted onto the most similar human germline antibodies from the databases. Select murine framework residues were retained based on knowledge of packing in the immunoglobulin fold (Chothia et al., 1998), the surface accessibility (Pedersen et al., 1994), the interface regions of VH/VL (Chothia et al., 1985), the Vernier zone (Foote and Winter, 1992) and other known risk factors (Studnicka et al., 1994). In total, four CDR-grafted designs were selected for each chain (
Following in-house design of both CoDAH and CDR grafted antibodies, a commercial service provider was contracted to synthesize, clone, express via transient transfection in HEK cells, and purify all constructs (MIGS LLC, Lebanon, N.H., USA). In addition to the five CoDAH designs with jointly optimized VH/Vκ pairs (
On average, CDR-grafted and CoDAH humanized antibody designs possessed similar levels of humanness. Their average HSC scores (CoDAH: 83.8 and grafted: 83.7) were similar across all the designs (
To facilitate comparison with traditional CDR grafting, the allowed mutations were restricted to those from a small set of the most-similar germline sequences: three for VH (IGHV4-4, 34 and 59) and two for Vκ (IGKV6D-21 and 41). Mutations were not allowed within the complementarity determining regions (CDRs), as defined by the Kabat numbering scheme using the AbNum program (Abhinandan and Martin, 2008). No further restrictions were imposed on allowed mutations.
Representative human germline sequences for IGHV4-4, IGHV4-34, IGHV4-59, IGKV6D-21, and IGKV6D-41 are shown below in Table 3.
The relative stability of all constructs, formatted as Fab fragments, was analyzed by differential scanning fluorimetry (DSF) (Niesen, Berglund and Vedadi, 2007) using an ABI 7500 Fast Real-Time PCR System from Applied Biosystems. Proteins and SYPRO Orange were diluted in PBS. Final protein concentrations were 100 μg/ml and final dye concentrations were 5×. The PCR gradient was run from 25-98° C. with a 1 min equilibration at each degree centigrade. Fluorescence was quantified using the preset TAMRA parameters. To eliminate confounding signals from Fc denaturation, thermostability was determined using recombinant Fab fragments. Since some of the Fabs displayed multi-transition melting curves, fractional melting temperatures were computed by analyzing local transitions, ignoring those that plateaued below arbitrarily chosen thresholds (i.e., indicating relatively minor unfolding). For example, TM20* was computed from the minimum intensity to the first local plateau exceeding 20% of the total fluorescence signal (ignoring both transitions below 20% and any above the first local transition, where observed). Values for TM10* and TM50* were likewise determined, while TM100* indicates the midpoint between the minimum and maximum intensities.
Despite the absence of the Fc region, the majority of the CDR-grafted constructs and one of the CoDAH constructs exhibited complex melting curves consisting of two or more transitions (superposition in
The EGFR binding affinities of all antibodies were estimated using biolayer interferometry on a ForteBio Octet Red instrument (ForteBio, Menlo Park, Calif., USA). Purified IgG samples at 12.5 μg/ml in phosphate buffered saline (PBS, 137 mM NaCl, 2.6 mM KCl, 10 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.4) were immobilized on protein A tips (ForteBio) and exposed to 100 nM recombinant, soluble his tagged human EGFR (Sino Biologicals, North Wales, Pa., USA) in PBS. Association and dissociation rates and equilibrium affinity were determined using software provided with the instrument. Samples were analyzed as technical duplicates in two to three independent experiments, estimated KD values were normalized to that of the cetuximab parental antibody, and normalized values were employed for relative ranking of antibody binding activity.
While thermostability analysis was performed on Fab fragments, affinities were measured with full length IgG antibodies. All 25 CoDAH designs (both Pareto optimal and cross-pairs) retained the parental cetuximab binding activity (0.7-1.5-fold,
Maintaining stability and binding affinity are critical parallel objectives of antibody humanization projects, but the dominant methods of humanization continue to be largely empirical, relying upon trial and error optimization that can produce widely variable performance outcomes. For example, a recent study using experimentally driven in vitro somatic hypermutation methods yielded a CDR-grafted cetuximab variant with 7° C. improved thermostability but 8-fold loss in binding affinity (McConnell et al., 2013, McConnell et al., 2014). Here, we have demonstrated that computationally humanized cetuximab variants consistently display a combination of increased humanness, substantially improved thermostability, and near wild type binding affinity. While CoDAH is most efficient when drawing upon a large repertoire of germline sequences, here mutable amino acids at each position were selected from only the three VH (IGHV4-4, 34 and 59) and two Vκ (IGKV6D-21 and 41) sequences used for comparative CDR-grafting experiments. Even when focused on the same set of antibody germline sequences and targeting largely similar positions (
We hypothesize that the multiple transitions observed in the melting curves for grafted designs may come from inter-domain interactions. Interestingly, the P9A mutation of the G2 and G3 VH domains is one of the evident differences among grafted designs (
It is known that modification of CDRs and their neighboring residues can detrimentally impact binding affinity (Wiens et al., 1998), and CoDAH constructs universally avoided mutations adjacent to CDRs. Likewise, most grafted constructs excluded CDR-adjacent mutations, with the exception of the G2 VH chain, in which substitutions were made at positions 58, 61, 63, and 64. The Kabat and Chothia numbering schemes employ different definitions for CDR positions, and in particular Kabat CDR-H2 is substantially longer (Kabat: residues 50-65, and Chothia: residues 52-56). For G2 VH, the mutated positions were adjacent to CDR-H2 by the Chothia numbering, yet within CDR-H2 by the Kabat (see
In conclusion, we have demonstrated that the structure-based computational antibody humanization method, CoDAH, is a cost-effective and reliable tool to directly generate humanized antibody variants with high stability and binding affinity. This tool may prove useful for antibody engineers seeking to quickly and effectively humanize antibodies derived from foreign hosts.
Additional embodiments are within the claims.
This application claims the benefit of U.S. provisional patent application No. 62/340,985, filed May 24, 2016, the contents of which is incorporated herein by reference in its entirety.
This invention was made with government support under R01 GM098977 awarded by National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/034175 | 5/24/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62340985 | May 2016 | US |
