Patent Application
20240383988
The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases (RTKs), directs proliferation and differentiation of many different tissues. See Wieduwilt M J & Moasser M M (2008) The epidermal growth factor receptor family: biology driving targeted therapeutics. Cell Mol Life Sci 65 (10): 1566-1584. Overexpression of EGFR occurs in numerous epithelial tumors and is associated with poor clinical outcome. See e.g., Aratani K, et al. (2017) Overexpression of EGFR as an Independent Prognostic Factor in Adenocarcinoma of the Esophagogastric Junction. Anticancer Res 37 (6): 3129-3135. Bethune G, Bethune D, Ridgway N, & Xu Z (2010) Epidermal growth factor receptor (EGFR) in lung cancer: an overview and update. J Thorac Dis 2 (1): 48-51; Hashmi A A, et al. (2018) Prognostic significance of epidermal growth factor receptor (EGFR) over expression in urothelial carcinoma of urinary bladder. BMC Urol 18 (1): 59; Li W, et al. (2018) Overexpression of Epidermal Growth Factor Receptor (EGFR) and HER-2 in Bladder Carcinoma and Its Association with Patients' Clinical Features. Med Sci Monit 24:7178-7185. To date, four anti-EGFR antibodies have been approved for human therapy and several others are in clinical trials. However, use of even the most popular therapeutic antibodies, Cetuximab (CTX) and Panitumumab (PTMB), is limited to a small subset of EGFR-positive tumors. See Garcia-Foncillas J, et al. (2019) Distinguishing Features of Cetuximab and Panitumumab in Colorectal Cancer and Other Solid Tumors. Front Oncol 9:849.
Described herein are EGFR binding antibodies. These antibodies bind to domain II and/or domain III of human EGFR. Also described are bispecific antibodies that are capable of binding both domain II and domain III of EGFR. Such antibodies advantageously expand the therapeutic utility of targeting EGFR beyond previous EGFR antibodies and allow inhibition of EGFR signaling in the “open” confirmation, which allows the antibodies to be more effective and effective against a broader range of cancers and tumors. Accordingly, also described herein are methods and uses of the antibodies in treating cancers and tumors.
In one aspect described herein is an antibody or antigen binding fragment thereof comprising: a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 17; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 18; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23 to 38; d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 6; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 7; wherein the antibody binds to EGFR domain II. In certain embodiments, the antibody or antigen binding fragment thereof is chimeric or humanized. In certain embodiments, the antibody or antigen binding fragment thereof is an IgG antibody. In certain embodiments, the antibody or antigen binding fragment thereof is a Fab, F(ab)2, a single-domain antibody, or a single chain variable fragment (scFv). In certain embodiments, the antibody or antigen binding fragment thereof is a multispecific antibody. In certain embodiments, the antibody or antigen binding fragment thereof is a bispecific antibody. In certain embodiments, the antibody or antigen binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 16 or 20; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2 or 3. In certain embodiments, the antibody or antigen binding fragment binds to EGFR with a KD of 50 nanomolar or less. In certain embodiments, the antibody or antigen binding fragment binds to EGFR with a KD of 10 nanomolar or less.
In another aspect described herein is an EGFR domain III binding antibody comprising: a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 52; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 53; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 54 to 60; d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 6; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 7; wherein the antibody binds to EGFR domain III. In certain embodiments, the antibody or antigen binding fragment thereof is chimeric or humanized. In certain embodiments, the antibody or antigen binding fragment thereof is an IgG antibody. In certain embodiments, the antibody or antigen binding fragment thereof is a Fab, F(ab)2, a single-domain antibody, or a single chain variable fragment (scFv). In certain embodiments, the antibody or antigen binding fragment thereof is a multispecific antibody. In certain embodiments, the antibody or antigen binding fragment thereof is a bispecific antibody. In certain embodiments, the antibody or antigen binding fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 51; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2 or 3. In certain embodiments, the antibody or antigen binding fragment binds to EGFR with a KD of 500 nanomolar or less. In certain embodiments, the antibody or antigen binding fragment binds to EGFR with a KD of 300 nanomolar or less.
Also described herein in one aspect is a bispecific antibody or antigen binding fragment thereof that binds to EGFR domain II and EGFR domain III. In certain embodiments, the bispecific antibody comprises an EGFR domain II binding moiety, wherein the EGFR domain II binding moiety comprises: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 17; (b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 18; (c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23 to 38; (d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; (e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 6; and/or (f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7. In certain embodiments, the bispecific antibody comprises an EGFR domain III binding moiety, wherein the EGFR domain III antigen binding moiety comprises: (a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 52; (b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 53; (c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 54 to 60; (d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; € a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 6; and/or (f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7. Also described herein, is a bispecific antibody or antigen binding fragment thereof comprising an EGFR domain II and an EGFR domain III binding moiety, wherein the EGFR domain II binding moiety comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 17; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 18; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23 to 38; d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 6; and f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7; and wherein the EGFR domain III antigen binding moiety comprises: g) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 52; h) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 53; i) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 54 to 60; j) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; k) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 6; and/or 1) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7. In certain embodiments, the bispecific antibody or antigen binding fragment thereof is chimeric or humanized. In certain embodiments, the bispecific antibody or antigen binding fragment thereof comprises an IgG constant region. In certain embodiments, the EGFR domain II binding moiety and the EGFR domain III binding moiety comprises a common immunoglobulin light chain, wherein the common immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2 or 3. In certain embodiments, the EGFR domain II binding moiety comprises an EGFR domain II binding immunoglobulin heavy chain, wherein the EGFR domain II binding immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 16 or 20. In certain embodiments, the EGFR domain III binding moiety comprises an EGFR domain III binding immunoglobulin heavy chain, wherein the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 51. In certain embodiments, the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain, comprise an IgG constant region; the common immunoglobulin light chain is associated with the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain; and the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain associate with each other. In certain embodiments, the IgG constant region of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain differ at one or more amino acid positions such that the heterodimerization of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain is favored. In certain embodiments, one of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain comprises a T366W substitution (EU numbering) and the other of the EGFR domain II binding immunoglobulin heavy chain or the EGFR domain III binding immunoglobulin heavy chain comprises a T366S/L368A/Y407V substitution (EU numbering), such that the heterodimerization of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain is favored. In certain embodiments, one of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain comprises one or more substitutions selected from Q347M, Y348F, T350D, T389W, L368M (EU numbering) and the other of the EGFR domain II binding immunoglobulin heavy chain or the EGFR domain III binding immunoglobulin heavy chain comprises one or more substitutions selected from S354I, E357L, K359S, T366S, L368A, and Y412V (EU numbering), such that the heterodimerization of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain is favored. In certain embodiments, one of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% or is 100% identical to SEQ ID NO: 61 and the other of the EGFR domain II binding immunoglobulin heavy chain or the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% or is 100% identical to SEQ ID NO: 62, such that the heterodimerization of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain is favored. In certain embodiments, the bispecific antibody or antigen binding fragment binds to EGFR with a KD of 100 nanomolar or less. In certain embodiments, the bispecific antibody or antigen binding fragment binds to EGFR with a KD of 50 nanomolar or less. In certain embodiments, the bispecific antibody or antigen binding fragment binds to EGFR with a KD of 10 nanomolar or less. In certain embodiments, the bispecific antibody or antigen binding fragment binds to the open confirmation of EGFR.
Also described herein is an antibody, bispecific antibody or antigen binding fragment thereof of that bind the same epitope or overlaps in binding with an antibody or bispecific antibody or antigen binding fragment thereof disclosed herein.
Also described herein is an antibody, bispecific antibody or antigen binding fragment thereof of that competes for binding with the antibody, bispecific antibody or antigen binding fragment thereof disclosed herein.
Also described herein is a composition comprising an antibody, bispecific antibody or antigen binding fragment thereof described herein and a pharmaceutically acceptable diluent, carrier, or excipient.
Also described herein is a nucleic acid or plurality of nucleic acids comprising a polynucleotide sequence encoding an antibody, bispecific antibody or antigen binding fragment thereof described herein.
Also described herein is a cell comprising the nucleic acid or plurality of nucleic acids comprising a polynucleotide sequence encoding an antibody, bispecific antibody or antigen binding fragment thereof described herein. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the cell is a prokaryotic cell.
Also described herein is the use of the antibody, bispecific antibody or antigen binding fragment thereof described herein in a method of treating cancer or tumor in an individual. In certain embodiments, the cancer or tumor is a solid tissue cancer or tumor. In certain embodiments, the cancer or tumor express EGFR. In certain embodiments, the cancer or tumor over expresses EGFR. In certain embodiments, the cancer or tumor comprises colorectal cancer. In certain embodiments, the cancer or tumor comprises head and neck cancer.
Also described herein is a method of treating an individual afflicted with a cancer or a tumor comprising administering to the individual afflicted with the cancer or the tumor the antibody, bispecific antibody or antigen binding fragment described herein, thereby treating the cancer or the tumor. In certain embodiments, the cancer or tumor is a solid tissue cancer or tumor. In certain embodiments, the cancer or tumor express EGFR. In certain embodiments, the cancer or tumor over expresses EGFR. In certain embodiments, the cancer or tumor comprises colorectal cancer. In certain embodiments, the cancer or tumor comprises head and neck cancer. In certain embodiments, the cancer or tumor comprises non-small cell lung cancer (NSCLC).
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
The terms “cancer” and “tumor” refer to the physiological condition in mammals characterized by deregulated cell growth.
The term “EGFR” or “Epidermal growth factor receptor” refers to the protein product of the EGFR gene. In certain embodiments, the EGFR gene is a human EGFR gene. In certain embodiments, the EGFR gene is a mammalian EGFR gene. In certain embodiments, the EGFR gene is a simian EGFR gene. In certain embodiments, the EGFR gene is a mouse EGFR gene. In certain embodiments, the EGFR gene is a rat EGFR gene. An exemplary Human EGFR sequence is in SEQ ID NO 1. EGFR proteins of the present disclosure include naturally occurring variants and naturally occurring post-translational modifications able to be bound by the antibodies described herein.
The EGFR ectodomain (ECD) is comprised of four structured domains and exists in at least two distinct conformations. The domains of EGFR comprise Domain I (amino acids 1-165), domain II (amino acids 165-310), domain III (amino acids 310-480), and domain IV (amino acids 480-620). In the absence of ligand and at low concentrations, domain II interacts with domain IV and tethers the ECD in a closed conformation. Upon ligand binding at an interface between domains III and IV, the ECD adapts a radically different open conformation, which frees a dimerization arm in domain II to interact with another EGFR or other ErbB family member to form active signaling dimers. See Dawson J P, et al. (2005). Epidermal growth factor receptor dimerization and activation require ligand-induced conformational changes in the dimer interface. Mol Cell Biol 25 (17): 7734-7742.
As used herein the term “individual,” “patient,” or “subject” refers to individuals diagnosed with, suspected of being afflicted with, or at-risk of developing at least one disease for which the described compositions and method are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In certain embodiments, the individual is a human.
As used herein, the term “about” used to modify a specific number refers to that number plus or minus 10% of that number. The term “about” modifying a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.
As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen used for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made. Skilled artisans will recognize that given a population of potential individuals for treatment not all will respond or respond equally to the treatment. Such individuals are considered treated if administered the compositions including the pharmaceutical compositions described herein.
The term “antibody” herein is used in the broadest sense and includes multivalent or bispecific antibodies and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. Antibodies as described herein also comprise multispecific or bispecific binding molecules that incorporate one or more complementarity determining regions that contribute to specific binding to a given target antigen. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. The antibody can comprise a human IgG1 constant region. The antibody can comprise a human IgG4 constant region.
Among the provided antibodies are multispecific or multivalent antibodies (for example, bispecific antibodies and polyreactive antibodies) and antibody fragments thereof. The antibodies include antibody-conjugates and molecules comprising the antibodies, such as chimeric molecules. Thus, an antibody includes, but is not limited to, full-length and native antibodies, as well as fragments and portion thereof retaining the binding specificities thereof, such as any specific binding portion thereof including those having any number of, immunoglobulin classes and/or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including but not limited to Fab, F(ab′)2, Fv, and scFv (single chain or related entity). A monoclonal antibody is generally one within a composition of substantially homogeneous antibodies; thus, any individual antibodies comprised within the monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in minor amounts. A monoclonal antibody can comprise a human IgG1 constant region or a human IgG4 constant region.
The terms “complementarity determining region,” and “CDR,” which are synonymous with “hypervariable region” or “HVR,” are known in the art and refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, M D (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme); Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan; 27 (1): 55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309 (3): 657-70, (“Aho” numbering scheme); and Whitelegg N R and Rees A R, “WAM: an improved algorithm for modelling antibodies on the WEB,” Protein Eng. 2000 Dec; 13 (12): 819-24 (“AbM” numbering scheme. In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.
The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs (See e.g., Kindt et al. Kuby Immunology, 6th ed., W. H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (See e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).
Among the provided antibodies are antibody fragments. An “antibody fragment” can refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2: diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv or sFv); and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., polypeptide linkers, and/or those that are not produced by enzyme digestion of a naturally-occurring intact antibody.
Herein a molecule, peptide, polypeptide, antibody, or antibody fragment can be referred to as “bispecific” or “dual-specific” including grammatical equivalents. A bispecific molecule possesses the ability to specifically bind to at least two structurally distinct targets. The specific binding may be the result of two distinct binding moieties that are structurally distinct at the molecular level, including but not limited to distinct non-identical amino acid sequences; or a single binding moiety that is able to specifically bind to two structurally distinct targets with high affinity (e.g., with a KD less than about 1×10−6). A molecule, peptide, polypeptide, antibody, or antibody fragment referred to as “multi-specific” refers to a molecule that possesses the ability to specifically bind to at least three structurally distinct targets. A “bispecific antibody” including grammatical equivalents refers to a bispecific molecule that preserves at least one fragment of an antibody able to specifically bind a target, for example, a variable region, heavy or light chain, or one or more complementarity determining regions from an antibody molecule. A “multi-specific antibody” including grammatical equivalents refers to a multi-specific molecule that preserves at least one fragment of an antibody able to specifically bind with a target, for example, a variable region, heavy or light chain, or complementarity determining region from an antibody molecule. A “common light chain bispecific antibody” refers to a specific type of bispecific antibody with two distinct heavy chains that can each form with the same light chain to bind to two structurally distinct targets.
“Fragment-based” bispecific antibodies or bispecific antibodies comprising a “single chain variable fragment” or “scFv” of this disclosure can refer to a single chain antibody, or fragment thereof, that comprises two binding moieties and a linker connecting the two binding moieties. The linker may be a polypeptide linker or other linker of suitable flexibility so as not to inhibit binding of either targeting moiety. Fragment based bispecific antibody formats include tandem VHH antibodies, tandem scFvs, scFv-Fabs, F(ab)2, dual-affinity retargeting antibodies (DARTs). Such fragment-based antibodies can be further manipulated to comprise additional binding moieties with specificity for a given target e.g., A2:B1, A1:B2 or A2:B2, or with fragments of an Fc region to improve pharmacokinetics or promote ADCC, ADCP, or CDC.
A “binding moiety” refers to a portion of a molecule, peptide, polypeptide, antibody, or antibody fragment that mediates specific binding to a recited target or antigen or epitope. By way of example, the binding moiety of an antibody may comprise a heavy-chain/light-chain variable region pair or one or more complementarity determining regions (CDRs).
Specific binding or binding of antibody molecules described herein refers to binding mediated by one or more CDR portions of the antibody. Not all CDRs may be required for specific binding. Specific binding can be demonstrated for example by an ELISA against a specific recited target or antigen that shows significant increase in binding compared to an isotype control antibody.
A “target” as referred to herein refers to the portion of a molecule that participates with a binding moiety of a molecule, peptide, polypeptide, antibody, or antibody fragment. A target can comprise an amino acid sequence and/or a carbohydrate, lipid or other chemical entity. An “antigen” is a target comprising a portion that is able to be bound by an adaptive immune molecule such as an antibody or antibody fragment, B-cell receptor, or T-cell receptor.
An “epitope” as described herein refers to the one or more contact regions of an antibody. The contact region of an antibody consists of a discreet number of amino acids contacted by amino acid residues of the antibody (generally CDR residues) and adjacent residues contiguous with the contact residues. For example, the contact region may consist of a continuous stretch of a target protein that is between 5 to 20 amino acids, 5 to 15 amino acids, or 5 to 10 amino acids. An antibody or bispecific antibody may bind more than one contact region that are separated by 10, 20, 30, 40, 50, 75, or 100 amino acids or more as a result of protein folding or multi-specificity of a bispecific antibody. Epitopes may be determined using X-ray crystallography, hydrogen-deuterium exchange mass-spec, alanine spanning mutagenesis, competition with excess synthetic peptides as determined by immunoblot, ELISA, surface plasmon radiance, flow cytometry or any other suitable protein binding assay.
As referred to herein “competition” can be determined by incubating a target with a first antibody or bispecific antibody with excess of a test antibody and observing a decrease in binding of the first antibody. Such decrease in binding can be observed using immunoblot, ELISA, surface plasmon radiance, flow cytometry or any other suitable protein binding assay.
The “valency” of a bispecific or multi-specific molecule refers to the number of targets a recited molecule, peptide, polypeptide, antibody, or antibody fragment is able to bind. For instance, a molecule that is monovalent is able to bind to one molecule of a specific target, a bivalent molecule is able to bind to two molecules, and a tetravalent molecule is able to bind four targets.
A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally can include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
Among the provided antibodies are human antibodies. A “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non-human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.
“ADCC” or “antibody dependent cell-mediated cytotoxicity” as used herein, refers to the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC can be correlated with binding to FcγRIIIa wherein increased binding to FcγRIIIa leads to an increase in ADCC activity. “ADCP” or antibody dependent cell-mediated phagocytosis, as used herein, can refer to the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.
The terms “polypeptide” and “protein” are used interchangeably and refers to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers and binding peptides, can include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides can contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
Amino acid sequence variants of the antibodies provided herein can be contemplated and conceived. A variant typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of known techniques. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. Antibody variants having one or more amino acid substitutions can be provided. Sites of interest for mutagenesis by substitution include the CDRs and FRs. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
The antibodies described herein can be encoded by a nucleic acid. A nucleic acid is a type of polynucleotide comprising two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer the polypeptide encoding polynucleotide into a cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a genomic integrated vector, or “integrated vector,” which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an “episomal” vector, e.g., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” Suitable vectors comprise plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors and the like. In the expression vectors regulatory elements such as promoters, enhancers, polyadenylation signals for use in controlling transcription can be derived from mammalian, microbial, viral or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and the like, may be employed. Plasmid vectors can be linearized for integration into a chromosomal location. Vectors can comprise sequences that direct site-specific integration into a defined location or restricted set of sites in the genome (e.g., AttP-AttB recombination). Additionally, vectors can comprise sequences derived from transposable elements.
As used herein, the terms “homologous,” “homology,” or “percent homology” when used herein to describe to an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.
The nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform, or otherwise render a suitable cell transgenic for the nucleic acid, thus enabling the production of antibodies for commercial or therapeutic uses. Standard cell lines and methods for the production of antibodies from a large-scale cell culture are known in the art. See e.g., Li et al., “Cell culture processes for monoclonal antibody production.” Mabs. 2010 Sep-Oct; 2 (5): 466-477. In certain embodiments, the cell is a Eukaryotic cell. In certain embodiments, the Eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a cell line useful for producing antibodies is a Chines Hamster Ovary cell (CHO) cell, an NS0 murine myeloma cell, or a PER.C6® cell. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of a cell useful for producing antibodies. In certain embodiments, described herein is a method of making an antibody comprising culturing a cell comprising a nucleic acid encoding an antibody under conditions in vitro sufficient to allow production and secretion of said antibody.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Described herein are antibodies that bind to and inhibit signaling through EGFR. Such antibodies are useful for the treatment for disorders that involve aberrant EGFR signaling. In some embodiments, the antibodies bind to EGFR domain II.
In certain embodiments, the EGFR antibodies and bispecific antibodies comprise Domain II binding moieties comprising CDR sequences of any one or more of antibodies 2a, 2b, or 2c described herein. In certain embodiments, the EGFR antibodies and bispecific antibodies comprise Domain III binding moieties comprising CDR sequences of any one or more of antibodies 3a, 3b, or 3c described herein.
In certain embodiments, described herein, is an EGFR domain II binding antibody comprising: a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 17; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 18; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23 to 38; d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 6; and f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 7.
In certain embodiments, the EGFR domain II binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that set forth in SEQ ID NO: 16 or 20; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 16 or 20; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 16 or 20; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 95% identical to that set forth in SEQ ID NO: 16 or 20; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 95% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 97% identical to that set forth in SEQ ID NO: 16 or 20; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 97% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 98% identical to that set forth in SEQ ID NO: 16 or 20; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 98% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 99% identical to that set forth in SEQ ID NO: 16 or 20; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 99% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence identical to that set forth in SEQ ID NO: 16 or 20; and wherein the immunoglobulin light chain comprises an amino acid sequence identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the domain II binding antibody or antigen binding fragment thereof is chimeric or humanized. In certain embodiments, the domain II binding antibody or antigen binding fragment thereof is an IgG antibody. In certain embodiments, the domain II binding antibody or antigen binding fragment thereof is a Fab, F(ab)2, a single-domain antibody, or a single chain variable fragment (scFv). In certain embodiments, the domain II binding antibody or antigen binding fragment thereof is a multispecific antibody. In certain embodiments, the domain II binding antibody or antigen binding fragment thereof is a bispecific antibody.
Described herein are antibodies that bind to and inhibit signaling through EGFR. Such antibodies are useful for the treatment for disorders that involve aberrant EGFR signaling. In some embodiments, the antibodies bind to EGFR domain III.
In certain embodiments, described herein, is an EGFR domain III binding antibody comprising: a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 52; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 53; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 54 to 60; d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 6; and f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 7.
In certain embodiments, the EGFR domain III binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that set forth in SEQ ID NO: 51; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain III binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 51; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain III binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 51; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain III binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 95% identical to that set forth in SEQ ID NO: 51; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 95% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain III binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 97% identical to that set forth in SEQ ID NO: 51; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 97% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain III binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 98% identical to that set forth in SEQ ID NO: 51; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 98% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain III binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 99% identical to that set forth in SEQ ID NO: 51; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 99% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain III binding antibody of the current disclosure comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises an amino acid sequence identical to that set forth in SEQ ID NO: 51; and wherein the immunoglobulin light chain comprises an amino acid sequence identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the domain III binding antibody or antigen binding fragment thereof is chimeric or humanized. In certain embodiments, the domain III binding antibody or antigen binding fragment thereof is an IgG antibody. In certain embodiments, the domain III binding antibody or antigen binding fragment thereof is a Fab, F(ab)2, a single-domain antibody, or a single chain variable fragment (scFv). In certain embodiments, the domain III binding antibody or antigen binding fragment thereof is a multispecific antibody. In certain embodiments, the domain III binding antibody or antigen binding fragment thereof is a bispecific antibody.
Provided herein are bispecific or multivalent antibody molecules comprising a first binding component configured to bind a first target and a second binding component configured to bind a second target, wherein the first target comprises an EGFR epitope, and wherein the second target comprises a distinct EGFR epitope. In certain embodiments, one binding moiety of the bispecific antibody binds to domain II of EGFR and the other binding moiety binds to domain III of EGFR. In certain embodiments, one binding moiety of the bispecific antibody binds to domain II of human EGFR and the other binding moiety binds to domain III of human EGFR. The bispecific molecule may be selective for the “open configuration” of EGFR (including human EGFR), distinguishing them from antibodies that bind the closed form such as Cetuximab or Panitumumab. Such bispecific antibodies are advantageous as they can inhibit EGFR activity in the presence of ligand or in the absence of ligand when EGFR can open due to a mass action effect as a result of high-expression levels.
A multispecific or bispecific antibody possesses the ability to specifically bind to at least two structurally distinct targets. The specific binding may be the result of two distinct binding moieties that are structurally distinct at the molecular level, including but not limited to distinct non-identical amino acid sequences; or a single binding moiety that is able to specifically bind to two structurally distinct targets. A molecule, peptide, polypeptide, antibody, or antibody fragment referred to as “multi-specific” or “multivalent” or “bispecific” can refer to a molecule that possesses the ability to specifically bind to at least two structurally distinct targets. In some embodiments, the first or the second binding component of the bispecific antibody comprises a polypeptide. In certain embodiments, the first or the second binding component consists of a polypeptide. In some embodiments, the first and the second binding component of the bispecific antibody comprises a polypeptide. In certain embodiments, the first and the second binding component consist of a polypeptide. In certain embodiments, the polypeptide of the first or second binding component comprises an amino acid sequence at least 100 amino acid residues in length. In certain embodiments, the polypeptide of the first and second binding component comprise an amino acid sequence at least 100 amino acid residues in length.
A bispecific molecule can be a bispecific antibody that preserves at least one fragment of an antibody able to specifically bind with a target, for example, a variable region, heavy or light chain, or one or more complementarity determining regions from an antibody molecule. In some embodiments, the antibody described herein is a bispecific antibody and/or dual antigen-binding fragment thereof. Bispecific antibodies possess the ability to bind to two structurally distinct targets or antigens. In some embodiments, the bispecific antibody comprises a first binding component configured to bind a first target and a second binding component configured to bind a second target, wherein the first target comprises EGFR domain II, and wherein the second target comprises an EGFR domain III.
The bispecific antibodies described herein reduce or eliminate signaling through EGFR. In certain embodiments, the bispecific antibodies inhibit signaling through the EGFR open confirmation. In certain embodiments, the binding affinity of the bispecific antibody when measured as KD is about 500 nanomolar or less, about 100 nanomolar or less, about 50 nanomolar or less, about 30 nanomolar or less, about 20 nanomolar or less, about 10 nanomolar or less.
Described herein, in certain embodiments, is a bispecific antibody or antigen binding fragment thereof comprising an EGFR domain II and an EGFR domain III binding moiety, wherein the EGFR domain II binding moiety comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 17; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 18; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23 to 38; d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 6; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7; and wherein the EGFR domain III antigen binding moiety comprises: g) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 52; h) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 53; i) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 54 to 60; j) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; k) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 6; and 1) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7. In certain embodiments, the bispecific antibody or antigen binding fragment thereof is chimeric or humanized. In certain embodiments, the bispecific antibody or antigen binding fragment thereof comprises an IgG constant region.
In certain embodiments, the EGFR domain II binding moiety and the EGFR domain III binding moiety comprises a common immunoglobulin light chain, wherein the common immunoglobulin light chain comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding moiety and the EGFR domain III binding moiety comprises a common immunoglobulin light chain, wherein the common immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding moiety and the EGFR domain III binding moiety comprises a common immunoglobulin light chain, wherein the common immunoglobulin light chain comprises an amino acid sequence at least about 95% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding moiety and the EGFR domain III binding moiety comprises a common immunoglobulin light chain, wherein the common immunoglobulin light chain comprises an amino acid sequence at least about 97% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding moiety and the EGFR domain III binding moiety comprises a common immunoglobulin light chain, wherein the common immunoglobulin light chain comprises an amino acid sequence at least about 99% identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding moiety and the EGFR domain III binding moiety comprises a common immunoglobulin light chain, wherein the common immunoglobulin light chain comprises an amino acid sequence identical to that set forth in SEQ ID NO: 2 or 3.
In certain embodiments, the EGFR domain II binding moiety comprises an EGFR domain II binding immunoglobulin heavy chain, wherein the EGFR domain II binding immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that set forth in SEQ ID NO: 16 or 20.
In certain embodiments, the EGFR domain II binding moiety comprises an EGFR domain II binding immunoglobulin heavy chain, wherein the EGFR domain II binding immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 16 or 20.
In certain embodiments, the EGFR domain II binding moiety comprises an EGFR domain II binding immunoglobulin heavy chain, wherein the EGFR domain II binding immunoglobulin heavy chain comprises an amino acid sequence at least about 95% identical to that set forth in SEQ ID NO: 16 or 20.
In certain embodiments, the EGFR domain II binding moiety comprises an EGFR domain II binding immunoglobulin heavy chain, wherein the EGFR domain II binding immunoglobulin heavy chain comprises an amino acid sequence at least about 97% identical to that set forth in SEQ ID NO: 16 or 20.
In certain embodiments, the EGFR domain II binding moiety comprises an EGFR domain II binding immunoglobulin heavy chain, wherein the EGFR domain II binding immunoglobulin heavy chain comprises an amino acid sequence at least about 98% identical to that set forth in SEQ ID NO: 16 or 20.
In certain embodiments, the EGFR domain II binding moiety comprises an EGFR domain II binding immunoglobulin heavy chain, wherein the EGFR domain II binding immunoglobulin heavy chain comprises an amino acid sequence at least about 99% identical to that set forth in SEQ ID NO: 16 or 20.
In certain embodiments, the EGFR domain II binding moiety comprises an EGFR domain II binding immunoglobulin heavy chain, wherein the EGFR domain II binding immunoglobulin heavy chain comprises an amino acid sequence identical to that set forth in SEQ ID NO: 16 or 20.
In certain embodiments, the EGFR domain III binding moiety comprises an EGFR domain III binding immunoglobulin heavy chain, wherein the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that set forth in SEQ ID NO: 51.
In certain embodiments, the EGFR domain III binding moiety comprises an EGFR domain III binding immunoglobulin heavy chain, wherein the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 51.
In certain embodiments, the EGFR domain III binding moiety comprises an EGFR domain III binding immunoglobulin heavy chain, wherein the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence at least about 95% identical to that set forth in SEQ ID NO: 51.
In certain embodiments, the EGFR domain III binding moiety comprises an EGFR domain III binding immunoglobulin heavy chain, wherein the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence at least about 97% identical to that set forth in SEQ ID NO: 51.
In certain embodiments, the EGFR domain III binding moiety comprises an EGFR domain III binding immunoglobulin heavy chain, wherein the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence at least about 98% identical to that set forth in SEQ ID NO: 51.
In certain embodiments, the EGFR domain III binding moiety comprises an EGFR domain III binding immunoglobulin heavy chain, wherein the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence at least about 99% identical to that set forth in SEQ ID NO: 51.
In certain embodiments, the EGFR domain III binding moiety comprises an EGFR domain III binding immunoglobulin heavy chain, wherein the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence identical to that set forth in SEQ ID NO: 51.
Disclosed herein are bispecific antibodies having a common light chain bispecific IgG structure, as shown in
In certain embodiments, the bispecific antibody or antigen binding fragment thereof comprises an EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain, comprising an IgG constant region; the common immunoglobulin light chain is associated with the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain; and the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain associate with each other. In certain embodiments, the IgG constant region of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain differ at one or more amino acid positions such that the heterodimerization of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain is favored. In certain embodiments, the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain comprises a T366W substitution (EU numbering) and the other of the EGFR domain II binding immunoglobulin heavy chain or the EGFR domain III binding immunoglobulin heavy chain comprises a T366S/L368A/Y407V substitution (EU numbering), such that the heterodimerization of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain is favored. In certain embodiments, the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain comprises one or more substitutions selected from Q347M, Y348F, T350D, T389W, L368M (EU numbering) and the other of the EGFR domain II binding immunoglobulin heavy chain or the EGFR domain III binding immunoglobulin heavy chain comprises one or more substitutions selected from S354I, E357L, K359S, T366S, L368A, and Y412V (EU numbering), such that the heterodimerization of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain is favored. In certain embodiments, the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% or is 100% identical to SEQ ID NO: 61 and the other of the EGFR domain II binding immunoglobulin heavy chain or the EGFR domain III binding immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% or is 100% identical to SEQ ID NO: 62, such that the heterodimerization of the EGFR domain II binding immunoglobulin heavy chain and the EGFR domain III binding immunoglobulin heavy chain is favored. In certain embodiments this sequence identity is maintained while preserving variant amino acids introduced to promote adequate homodimerization.
Compositions comprising the antibodies of the current disclosure are included in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. In certain embodiments, the antibodies of the current disclosure are administered suspended in a sterile and/or isotonic solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of: buffers, for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris); surfactants, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; polyol/disaccharide/polysaccharides, for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, for example, glycine or arginine; antioxidants, for example, ascorbic acid, methionine; or chelating agents, for example, EDTA or EGTA.
Subcutaneous formulations for administration of antibodies can comprise one or more of: buffers, for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris); surfactants, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; polyol/disaccharide/polysaccharides, for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, for example, glycine or arginine; antioxidants, for example, ascorbic acid, methionine; or chelating agents, for example, EDTA or EGTA. Additionally, a compound or molecule that relieves pain at the injection site can be included, such as hyaluronidase, for example at a concentration of from about 2,000 U/ml to about 12,000 U/ml.
In certain embodiments, the antibodies or bispecific antibodies of the current disclosure are shipped/stored lyophilized and reconstituted before administration. In certain embodiments, lyophilized antibody formulations comprise a bulking agent such as, mannitol, sorbitol, sucrose, trehalose, dextran 40, or combinations thereof. The lyophilized formulation can be contained in a vial comprised of glass or other suitable non-reactive material. The antibodies when formulated, whether reconstituted or not, can be buffered at a certain pH, generally less than about 7.5. In certain embodiments, the pH can be between 4.5 and 7.5, 4.5 and 7.0, 4.5 and 6.5, 4.5 and 6.0, or 5.5 or 5.0.
Also described herein are kits comprising one or more of the antibody molecules described herein in a suitable container and one or more additional components selected from: instructions for use; a diluent, an excipient, a carrier, and a device for administration.
In certain embodiments, described herein is a method of preparing a cancer treatment comprising admixing one or more pharmaceutically acceptable excipients, carriers, or diluents and an antibody molecule of the current disclosure. In certain embodiments, described herein is a method of preparing a cancer treatment for storage or shipping comprising lyophilizing one or more antibodies of the current disclosure.
The nucleic acids encoding the antibodies or bispecific antibodies described herein can be used to infect, transfect, transform, or otherwise render a suitable cell transgenic for the nucleic acid, thus enabling the production of antibodies or bispecific antibodies for commercial or therapeutic uses. Standard cell lines and methods for the production of antibodies from a large-scale cell culture are known in the art. See e.g., Li et al., “Cell culture processes for monoclonal antibody production.” Mabs. 2010 Sep-Oct; 2 (5): 466-477.
In certain embodiments, a nucleic acid sequence encodes the antibodies or bispecific antibodies disclosed herein. In certain embodiments, the polynucleotide sequence encoding the antibodies or bispecific antibodies is operatively coupled to a eukaryotic regulatory sequence. In some embodiments, a cell comprises the nucleic acid sequence.
In some embodiments, a cell comprises a nucleic acid encoding the antibodies or bispecific antibodies disclosed herein. In certain embodiments, the cell comprises a prokaryotic cell. In certain embodiments, the prokaryotic cell is an Escherichia coli cell. In certain embodiments, the cell comprises a eukaryotic cell. In certain embodiments, the eukaryotic cell is a Chines Hamster Ovary (CHO) cell, an NS0 murine myeloma cell, or a human PER.C6 cell.
In certain embodiments, described herein is a method of making antibodies or bispecific antibodies comprising culturing a cell comprising a nucleic acid encoding a antibodies or bispecific antibodies under conditions in vitro sufficient to allow production and secretion of the antibodies or bispecific antibodies.
In certain embodiments, described herein, is a master cell bank comprising: (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol. In certain embodiments, the master cell bank comprises: (a) a CHO cell line comprising a nucleic acid encoding the antibodies or bispecific antibodies integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO or both. In certain embodiments, the master cell bank is contained in a suitable vial or container able to withstand freezing by liquid nitrogen.
Also described herein are methods of making antibodies or bispecific antibodies described herein. Such methods comprise incubating a cell or cell-line comprising a nucleic acid encoding the antibodies or bispecific antibodies in a cell culture medium under conditions sufficient to allow for expression and secretion of the antibodies or bispecific antibodies, and further harvesting the antibodies or bispecific antibodies from the cell culture medium. The harvesting can further comprise one or more purification steps to remove live cells, cellular debris, non-antibody proteins or polypeptides, undesired salts, buffers, and medium components. In certain embodiments, the additional purification step(s) include centrifugation, ultracentrifugation, protein A, protein G, protein A/G, or protein L purification, and/or ion exchange chromatography.
In certain embodiments, disclosed herein, are bispecific antibodies useful for the treatment of a cancer or tumor. Treatment refers to a method that seeks to improve or ameliorate the condition being treated. With respect to cancer, treatment includes, but is not limited to, reduction of tumor volume, reduction in growth of tumor volume, increase in progression-free survival, or overall life expectancy. In certain embodiments, treatment will affect remission of a cancer being treated. In certain embodiments, treatment encompasses use as a prophylactic or maintenance dose intended to prevent reoccurrence or progression of a previously treated cancer or tumor. It is understood by those of skill in the art that not all individuals will respond equally or at all to a treatment that is administered, nevertheless these individuals are considered to be treated.
In certain embodiments, the cancer or tumor is a solid tissue cancer or tumor. In certain embodiments, the cancer or tumor is a blood cancer. In certain embodiments, the cancer is metastatic. In certain embodiments, the cancer is one associated with aberrant EGFR expression, such as over expression. In certain embodiments, the cancer or tumor is colorectal cancer. In certain embodiments, the cancer or tumor is metastatic colorectal cancer. In certain embodiments, the cancer or tumor is head and neck cancer. In certain embodiments, the cancer or tumor is metastatic head and neck cancer. In certain embodiments, the cancer or tumor comprises non-small cell lung cancer (NSCLC).
In certain embodiments, the antibodies and bispecific antibodies described can be administered to a subject in need thereof by any route suitable for the administration of antibody-containing pharmaceutical compositions, such as, for example, subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral, or intracerebral, etc. In certain embodiments, the antibodies are administered intravenously. In certain embodiments, the antibodies are administered subcutaneously. In certain embodiments, the antibodies are administered intratumoral. In certain embodiments, the antibodies are administered on a suitable dosage schedule, for example, weekly, twice weekly, monthly, twice monthly, once every two weeks, once every three weeks, or once a month etc. The antibodies can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1 mg/kg and about 50 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg/kg and about 40 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg/kg and about 30 mg/kg. Therapeutically effective amounts include amounts that are sufficient to ameliorate one or more symptoms associated with the disease or affliction to be treated.
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
To facilitate the production of Abs that could be efficiently developed as potential therapeutics, a phage-displayed library of antigen-binding fragments (Fabs) using the framework of anti-ErbB2 therapeutic Ab Trastuzumab was constructed. The light chain sequence of the library was fixed as that of a Trastuzumab variant and the three heavy chain complementarity-determining regions (CDRs) were diversified (
Following multiple rounds of selection for binding to the EGFR ectodomain (ECD), a number of antibodies capable of binding to EGFR as phage-Fab containing the LC variant were isolated and as IgGs produced with the wild-type Trastuzumab kappa light chain.
For characterization, focus was directed to two distinct anti-EGFR antibodies-named ER-2 and ER-3 which bound or did not bind to EGFR in the presence of EGF, respectively. Surface plasmon resonance (SPR) assays with purified IgG proteins revealed that ER-2 and ER-3 bound to EGFR with moderate affinities (KD)=20 or 14 nM, respectively, (
ELISA was also used to determine whether saturating concentrations of EGF or various anti-EGFR IgGs could block binding of each Fab to immobilized EGFR (
To map epitopes, flow cytometry was used to assess the binding of IgGs to HEK293T cells transfected to express full-length EGFR or fragments of EGFR comprising double domains (D1-D2, D2-D3, D3-D4) or triple domains (D1-D2-D3, D2-D3-D4), and plotted binding signals at saturating IgG concentrations (
Finally, the effects of the Abs on the activation of EGFR by EGF in MDA-MB-468 breast cancer cells was investigated, which display high levels of endogenous EGFR. See Filmus J, Pollak M N, Cailleau R, & Buick R N (1985) MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited by EGF. Biochem Biophys Res Commun 128 (2): 898-905. Western blotting revealed that both Fabs reduced levels of phosphorylated EGFR in a dose-dependent manner, and Fab ER-2a was particularly potent in this assay (
To improve the biophysical properties of IgGs ER-2a and ER-3a, focused phage displayed libraries that diversified the sequence of each heavy chain in a controlled and limited manner were constructed, and following selection and ELISA screening for binding to EGFR, a range of variants were identified including the optimized variants of ER-2a and ER-3a, which we named ER-2b and ER-3b, respectively (
A bispecific antibody was made incorporating the binding domains from the ER-3a and ER2a clones. As shown in
As shown in
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
PYYSYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYFHSSW
YESSSPRAYYPGMDYWGQGTLVTVSS
PYYSYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYYHISW
PWSSYGGTYYPGMDYWGQGTLVTVSS
PSSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARHWYYYP
PSSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARHWHVYP
SYYWGGFDYWGQGTLVTVSS
SYYWGGMDYWGQGTLVTVSS
SYYWGGMDYWGQGTLVTVSS
This application claims the benefit of U.S. Provisional Ser. No. 63/214,596 filed on Jun. 24, 2021, which is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/034990 | 6/24/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63214596 | Jun 2021 | US |
