Patent Application
20210332145
The instant application contains a Sequence Listing that is filed herewith by electronic submission and is hereby incorporated by reference in its entirety. The ASCII copy, created on 8 Oct. 2019 is named SCT0004-401-PC_Sequence_Listing_ST25.txt, and is 54,800 bytes in size.
B-cells are lymphocytes that play major roles in adaptive humoral immunity and production of antibodies that specifically recognize antigens. Three subclasses of B-cells are naïve B-cells, memory B-cells, and plasma cells. The processes of VDJ recombination, in which two or three segments of DNA are chosen from a genomic library and recombined to generate a combinatorial array of antibody variable domains, and hypermutation, by which the variable domains encoded by different lineages of B-cells are further varied, result in up to 109 distinct B-cell lineages that produce antibodies with specificity for distinct targets. A B-cell is said to be specific for an antigen that binds the antibodies made by that B-cell. B-cells in general are stimulated by exposure to their specific antigen (Ag). Naïve B-cells have not yet been exposed to their specific antigen. Such exposure (e.g., during an infection) results in proliferation of B-cells and generation of sister clones. Sister clones can develop into plasma cells, which produce high amounts of antibody. Plasma cells may either be short lived, or may migrate into bone marrow, where they can persist for an extended period of time. A sister clone of an Ag-exposed B-cell may also develop into a memory B-cell that is quiescent until re-exposed to the specific antigen. Memory B-cells respond rapidly to re-exposure to antigen by dividing to produce both plasma cells and additional memory B-cells.
Several significant diseases involve B-cells. Malignant transformation of B-cells leads to cancers, including some lymphomas such as, for example, multiple myeloma and Hodgkin's Lymphoma. Some autoimmune diseases, including systemic lupus erythematosus (SLE), also involve B-cells. Both cancer and autoimmune diseases that involve B-cells may be considered gain of function conditions, in that the B-cells overgrow and/or attack parts of the body inappropriately. A possible strategy to control such diseases is to use antibodies that target the pathological B-cells.
The B-cell Maturation Antigen (BCMA, also known as TNFRSF17 and CD269) is a protein that has been shown to be expressed on the surface of plasmablasts (i.e., plasma cell precursors) and plasma cells, and is believed to stimulate survival. It therefore represents a potential target for B-cell-related diseases. BCMA is a member of the TNF receptor family and binds the TNF family ligands BAFF and APRIL (Kalled et al., 2005). BCMA is a type III membrane protein, as it lacks the signal peptide associated with type I membrane proteins found in most TNF receptor family members.
The mechanism of action of BCMA is not fully understood. Mice that have been genetically altered to lack a functional gene for BCMA have normal lymphoid organs and cell populations, and a nearly normal functioning immune system (Xu and Lam, 2001; Schiemann et al., 2001). The only defect defined to date in these mice is a diminished survival of long-lived bone marrow (BM) plasma cells (O'Connor et al., 2004). Therefore, it may be that BCMA, at least in the murine system, provides a survival signal to BM-resident plasma cells that is either BAFF or APRIL-mediated, or both. Indeed, signaling through BCMA activates the NF-κB pathway (Hatzoglou et al., 2000) which is involved in B-cell survival, proliferation and maturation (Litinskiy et al., 2002; Pomerantz and Baltimore, 2002; Huang et al., 2004; He et al., 2004). Results with malignant human cells are generally consistent with a link between BCMA and cell survival. Primary multiple myeloma (MM) cells, MM cell lines (Novak et al., 2004a), and Hodgkin and Reed-Sternberg (HRS) cells from Hodgkin lymphomas (Chiu et al., 2007; Novak et al., 2004b) have been shown to express BCMA. Addition of BAFF and/or APRIL has further been shown to provide a survival signal for these malignant cells, although it is not clear that BCMA is predominantly responsible for this effect.
Because different B-cell subsets are implicated in different B-cell related conditions, there exists a need for agents that specifically target one or more B-cell subsets. The expression of BCMA on the surface of some B-cells provides a marker by which those cells may be specifically targeted. To take advantage of BCMA as a marker of one or more B-cell subsets, there is a need for agents that specifically bind to BCMA. The disclosure provides antibodies that specifically bind to BCMA. The antibodies of the disclosure may be used to target one or more of the following B-cell subsets: plasma cells, memory B-cells, and naïve B-cells, for therapeutic applications.
The disclosure provides antibodies that bind to BCMA and methods of use thereof. In one embodiment, the disclosure provides an isolated antibody that binds to SEQ ID NO:1.
The disclosure provides antibodies that bind specifically to SEQ ID NO:1. The term “antibody” as used herein, includes both full-length immunoglobulins and antibody fragments that bind to BCMA. The antibodies can be, e.g., a monoclonal, polyclonal, chimeric, humanized, or single chain antibody. As used herein, the terms “antigen binding fragment,” “fragment,” and “antibody fragment” are used interchangeably to refer to any fragment that comprises a portion of a full-length antibody, generally at least the antigen binding portion or the variable region thereof. Examples of antibody fragments include, but are not limited to, diabodies, single-chain antibody molecules, multispecific antibodies, Fab, Fab′, F(ab′)2, Fv or scFv.
The phrase “therapeutically effective” is intended to qualify the amount of active ingredients used in the treatment of a disease or disorder or on the effecting of a clinical endpoint.
The term “therapeutically acceptable” refers to those compounds (or salts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitable for use in contact with the tissues of patients without undue toxicity, irritation, and allergic response, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.
The terms “treating,” “treatment,” and the like, as used herein, mean ameliorating a disease, so as to reduce, ameliorate, or eliminate its cause, its progression, its severity, or one or more of its symptoms, or otherwise beneficially alter the disease in a subject. Reference to “treating,” or “treatment” of a patient is intended to include prophylaxis. Treatment may also be preemptive in nature, i.e., it may include prevention of disease in a subject exposed to or at risk for the disease. Prevention of a disease may involve complete protection from disease, for example as in the case of prevention of infection with a pathogen, or may involve prevention of disease progression. For example, prevention of a disease may not mean complete foreclosure of any effect related to the diseases at any level, but instead may mean prevention of the symptoms of a disease to a clinically significant or detectable level. Prevention of diseases may also mean prevention of progression of a disease to a later stage of the disease.
The terms “subject” and “patient” are used interchangeably herein to mean all mammals including humans. Examples of subjects include, but are not limited to, humans, monkeys, dogs, cats, horses, cows, goats, sheep, pigs, and rabbits. In one embodiment, the subject or patient is a human.
In some embodiments, the antibodies or antibody fragments of the present disclosure use BCMA to “target” B-cell lymphomas. In essence, such targeting can be generalized as follows: antibodies or antibody fragments of the present disclosure specific to the BCMA surface antigen of B-cells are, for example, injected into a subject and specifically bind to the BCMA cell surface antigen of both normal and malignant B-cells; this binding leads to the destruction and/or depletion of neoplastic B-cells. Additionally, chemical agents (e.g., chemotherapeutics) or radioactive labels (e.g., radiotherapeutics) having the potential to destroy cancer cells and/or tumors can be conjugated to the antibodies or antibody fragments of the present disclosure such that the agent is specifically “delivered” to the targeted B-cells, such as, for example, neoplastic B-cells. In some embodiments, the methods of the present disclosure comprise administering an antibody or antibody fragment that is not conjugated to a chemical agent or radioactive label. In some embodiments, the methods of the present disclosure comprise administering an antibody or antibody fragment that is not conjugated to a cytotoxic agent. For discovering and developing antibody therapeutics generally known in the art, please see U.S. Pat. No. 10,072,088, which is incorporated in its entirety by reference.
The provided antibodies may be used to diagnose, treat, or monitor BCMA-expressing cancer and its progression, regression, or stability; to determine whether or not a patient should be treated for cancer; or to determine whether or not a subject is afflicted with BCMA-expressing cancer and thus may be amenable to treatment with a BCMA-specific anti-cancer therapeutic. In some embodiments, the antibodies or fragments thereof described herein may be used for various in vitro molecular-biology applications such as, for example, enzyme-linked immunosorbent assays (ELISA), Western blots, immunohistochemistry, immunocytochemistry, flow cytometry and fluorescence-activated cell sorting (FACS), immunoprecipitation, and/or enzyme-linked immunespotting. In some embodiments, the antibodies or fragments thereof may be packaged in kits with or without additional reagents known to those of skill in the art for practicing any of the molecular biology techniques disclosed above.
The disclosure provides the antibodies SCT-Aa01, SCT-Aa02, SCT-Aa03, SCT-Aa04, SCT-Aa05, SCT-Aa06, SCT-Aa07, SCT-Aa08, SCT-Aa09, SCT-Aa10, SCT-Aa11, SCT-Aa12, SCT-Aa13, SCT-Aa14, SCT-Aa15, SCT-Aa16, SCT-Aa17, SCT-Aa18, and SCT-Aa19. Each of these is a murine monoclonal antibody.
Additionally, recombinant anti-BCMA antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the disclosure. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques such as, for example, the methods described in U.S. Pat. No. 7,112,421; Better et al. (1988) Science 240:1041-1043; or Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443.
The antibodies of the disclosure may comprise the heavy chain variable domain sequences of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39. The heavy chain variable domain sequences may consist essentially of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39.
The antibodies of the disclosure may comprise the light chain variable domain sequences of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:19, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. The light chain variable domain sequences may consist essentially of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11 SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:19, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40.
The disclosure also provides a variable domain sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to a sequence selected from SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39. The disclosure also provides a variable domain sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to a sequence selected from SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11 SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40. The disclosure also provides antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:4 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:5. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:6 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:7. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:8 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:9. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:10 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:11. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:12 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:13. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:14 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:15. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:16 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:17. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:18 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:19. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:20 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:21. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:22 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:23. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:24 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:25. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:26 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:27. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:28 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:19. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:29 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:30. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:31 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:32. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:33 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:34. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:35 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:36. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:37 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:38. The disclosure includes antibodies comprising a heavy chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:39 and a light chain variable domain sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:40.
Table 1 provides a summary of the BCMA-specific antibodies described herein.
Pharmaceutical compositions comprising the antibodies or antibody fragments of the present disclosure are also contemplated and can be used in the methods disclosed herein. Pharmaceutical compositions can comprise one or more of the antibodies or antibody fragments described herein and a pharmaceutically acceptable carrier or excipient. Although the carrier or excipient may facilitate administration, it should not itself induce the production of antibodies harmful to the subject or individual receiving the composition; nor should it be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles, and are known to one of skill in the art.
The antibodies or an antigen binding fragments described herein, or the pharmaceutical compositions disclosed herein, may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal or rectal routes. Typically, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
In one embodiment, the antibody, or an antigen binding fragment thereof, or pharmaceutical composition is administered intravenously. In another embodiment, the antibody, or an antigen binding fragment thereof, or pharmaceutical composition is administered by intravenous infusion.
Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule. Known antibody-based pharmaceuticals provide guidance relating to frequency of administration e.g., whether a pharmaceutical should be delivered daily, weekly, monthly, etc. Frequency and dosage may also depend on the severity of symptoms.
It will be appreciated that the active ingredient in the composition will be an antibody molecule, an antibody fragment or variants and derivatives thereof. As such, it will be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition will need to contain agents which protect the antibody from degradation but which release the antibody once it has been absorbed from the gastrointestinal tract.
The methods of the present invention can use an antibody, or an antigen binding fragment thereof, as described above, alone or in combination with other pharmaceutically active compounds, to treat conditions such as those disclosed hereinabove. The additional pharmaceutically active compound(s) can be administered simultaneously (either in the same dosage form or in separate dosage forms) or sequentially. Accordingly, in one embodiment, the present invention comprises methods for treating a condition by administering to the subject a therapeutically-effective amount of an antibody, or an antigen binding fragment thereof, of the present invention and one or more additional pharmaceutically active compounds.
In some embodiments, the antibody, or an antigen binding fragment thereof, of the present invention is used in combination with existing BCMA-related disease therapies.
The following example is put forth so as to provide those of ordinary skill in the art with a complete description of how to make and use the present disclosure and is not intended to limit the scope of present disclosure nor is it intended to represent that the experiment below is all or the only experiment that could be performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Recombinant human BCMA (huBCMA-His, Catalog No.: BCA-H522y, ACROBiosystems, Beijing, China, SEQ ID NO:2) was used to immunize young C57/BL6 mice each with 80 μg huBCMA-His in Sigma Adjuvant System® (Sigma-Aldrich, St. Louis, Mo.) using a rapid immunization protocol of Antibody Solutions (Sunnyvale, Calif.). The lymph nodes were harvested along with serum on day 35. Single cell suspension of the lymph node was generated, and the suspension was filtered through a 70 μm mesh (BD Bioscience) to remove clumps.
The filtered lymphocyte suspension was enriched for plasma cells actively secreting IgGs instead of using a kit based on cell surface expression of CD138 (Miltenyi, Auburn, Calif.). Afterwards, freshly enriched plasma cells were spread on a PDMS device to allow a single cell deposition in the microwells on the device. Antibody secreted from each ASC was captured on a derivatized microscope slide (U.S. Pat. No. 9,328,172). Antigen-specific antibody secreting cells were identified by interrogating the antibody capture slide with varying concentrations of fluorescently labeled BCMA-huFC (ACROBiosystems, Beijing, China, Catalog No.: BC7-H5254, SEQ ID NO:3).
mRNA Capture
After antibody capture, the medium was removed, and replaced with lysis buffer followed by prompt closure of the top of the microwells with a custom oligonucleotide microarray on a microscope slide (Agilent, Santa Clara, Calif.). This procedure was previously described in U.S. Pat. No. 9,328,172. The custom oligonucleotide microarray is prepared such that each feature contains not only a unique tag specifying its coordinate but also capture probes for all subclasses (1, 2a, 2b, and 3) of murine IgG heavy chain, murine Ig kappa light chain. Hybridization was allowed to proceed overnight.
cDNA Synthesis, PCR Amplification, and Next Generation Sequencing
Captured mRNA on the custom microarray was further processed to synthesize cDNA of each mRNA incorporating the unique tag originally on each feature. The cDNA is then amplify using a Taq polymerase (Promega, Madison, Wis.) and appropriate set of primers to allow amplification of the following genes: variable domain of IgG heavy chain subclasses and variable domain of Ig kappa light chain. Though now released from cells, these fragments of each gene are now labeled with the unique tag from the custom oligonucleotide microarray manifesting their originating locations. The amplicons were further manipulated to have appropriate sequence attached at both ends to enable sequencing on an Illumina MiSeq instrument using 2×250 bp chemistry at SeqMatic LLC (Fremont, Calif.).
Sequencing reads from MiSeq were processed and the embedded tag in each read was identified and converted into coordinates. The coordinates were plotted to yield a synthetic map of the mRNA recovered. Most of the coordinates form clusters that designate the location of the originating cell for the recovered mRNA sequences. Next, a complementarity-determining region 3 (CDR3) motif present in each read with the coordinates was identified and collated according to the clusters that matched the location of an antibody spot visualized by an appropriate fluorescently labeled secondary antibody. Identical or nearly identical CDR3s for a given antibody spot were organized and form consistent pair of VH and VL sequences. The remaining part of sequences containing the identified CDR3s were identified and assembled into full-length cDNA sequences for VH and VL. The pair of full-length cDNA was correlated with the affinity measurements associated with each of the antigen-specific antibody spot.
The paired VH and VL anti-BCMA antibody sequences were used to synthesize corresponding gene fragments by a service provider according to the known art. The resulting gene fragments were each cloned into an appropriate plasmid vector with either mouse IgG1 heavy chain constant region and mouse kappa light chain constant region or human IgG1 heavy chain constant region and human kappa light chain and the appropriate H and L chain pair was transfected into an appropriate mammalian host, such as HEK293, for recombinant expression to produce an antibody preparation in full-IgG format. The antibody preparations were characterized by measurements at OD280 to assess the amount produced and by gel electrophoresis on PAGE to assess the size of the antibody chains produced. A subset of the anti-human BCMA antibodies of the present disclosure was reconstructed and recombinantly expressed. Their respective clone preparation IDs are shown in Table 2.
The recombinantly expressed antibodies were used to assess binding activity to BCMA of different species origins by conventional ELISA according to the known art. Recombinant human BCMA, cyno BCMA, and mouse BCMA were coated onto ELISA plates to detect binding at serially diluted concentrations of the anti-BCMA antibody preparations.
The ability for antibodies of the present disclosure to bind human BCMA was measured with an ELISA assay. For the BCMA binding assay, a 96-well plate (Nunc) is coated with human BCMA with a His tag (ACROBiosystems, Catalog No.: AC108P1-180611F2-Bulk, SEQ ID NO:2) at 0.2 μg/well overnight at 4° C. Wells are blocked for 2 h with blocking buffer (PBS containing 3% bovine serum albumin). Wells are washed three times with PBS containing 0.1% Tween-20. Anti-human BCMA antibodies of the present disclosure or control IgG (100 μL) is then added and incubated at room temperature for 1 h. After washing, the plate is incubated with 100 μL of goat anti-mouse IgG Fcγ Fragment Specific-HRP conjugate (Jackson ImmunoResearch) at room temperature for 1 h. The plates are washed and then incubated with 100 μL of 3,3′,5,5′-tetra-methylbenzidine. The absorbance at 450 nm is read on a microplate reader. The half maximal effective concentration (EC50) is calculated using GraphPad Prism 6 software.
In experiments performed essentially as described above, anti-human BCMA antibodies of the present disclosure bind human BCMA with a His tag (ACROBiosystems, Beijing, China, Catalog No.: BCA-H522y, SEQ ID NO:2) with an EC50 values shown in Table 3.
In experiments performed essentially as described above, anti-human BCMA antibodies of the present disclosure bind cyno BCMA with a His tag (ACROBiosystems, Beijing, China, Catalog No.: BCA-052H7, SEQ ID NO:110) with an EC50 values shown in Table 4.
In experiments performed essentially as described above, anti-human BCMA antibodies of the present disclosure bind mouse BCMA with a His tag (ACROBiosystems, Beijing, China, Catalog No.: BCA-M52H3, SEQ ID NO:109) with an EC50 values shown in Table 5.
The binding affinity of select anti-BCMA was measured on a surface plasmon resonance (SPR) instrument, Biacore T200™, against human BCMA by a method known in the art.
In experiments performed essentially as described above, a subset of anti-human BCMA antibodies of the present disclosure bind human BCMA with a His tag (ACROBiosystems, Beijing, China, Catalog No.: BCA-H522y, SEQ ID NO:2) with affinity shown in Table 6.
A subset of anti-human BCMA antibodies of the present disclosure were assayed for binding to 293T cells transfected with BCMA and the flow cytometry plot for one of the antibodies (SCT-Aa02) is shown in
A subset of anti-human BCMA antibodies of the present disclosure were assayed for binding to 293T cells transfected with BCMA and the fraction of cells bound the labeled antibody above background defined by control with parental 293T cells is shown in Table 7.
A cell line expressing BCMA, H929, was used to assess binding by anti-BCMA antibodies on the native protein on the cell surface by flow cytometry according to the known art. A subset of anti-human BCMA antibodies were assayed for binding to H929 and the fraction of cells bound the labeled antibody above background defined by control is shown in Table 8.
In addition, ADCC activity of a subset of anti-BCMA antibodies was assessed using an engineered surrogate effector cell line, kit reagents, and a luminometer (Promega, Madison, Wis.) on 2 cell lines expressing BCMA, MM.1S and U266, as target cells and the results were used to compare against two benchmark antibodies (chA7D12.2 and chC12A3.2) from U.S. Pat. No. 9,034,324 with known ADCC activity (
Some of the antibodies recovered from the antibody campaign described above are listed herein. The complementarity-determining region (CDR) sequences for the anti-BCMA antibodies heavy-chains (e.g., HCDR1) and light-chains (e.g., LCDR1) described herein are depicted in Table 11. The heavy-chain variable domain (VH) and light-chain variable domain (VL) sequences for the anti-BCMA antibodies described herein are depicted in Table 12.
The preceding merely illustrates the principles of the disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the disclosure and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles and concepts of the disclosure, further the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the disclosure as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present disclosure, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present disclosure is embodied by the appended claims.
This application claims priority to U.S. Provisional Application No. 62/743,413 filed 9 Oct. 2018, which is incorporated herein by reference in its entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/110114 | 10/9/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62743413 | Oct 2018 | US |
