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WO2025130984A1 - Single domain antibodies for bcma - Google Patents

Single domain antibodies for bcma Download PDF

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Publication number
WO2025130984A1
WO2025130984A1 PCT/CN2024/140658 CN2024140658W WO2025130984A1 WO 2025130984 A1 WO2025130984 A1 WO 2025130984A1 CN 2024140658 W CN2024140658 W CN 2024140658W WO 2025130984 A1 WO2025130984 A1 WO 2025130984A1
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seq
antibody
amino acid
acid sequence
antibodies
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French (fr)
Inventor
Yonghua Luo
Weijie LAN
Weimin Li
Hongtao Li
Yunfeng Eric HU
Sihui GUAN
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Overland Therapeutics Us Inc
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Overland Therapeutics Us Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • BCMA B-cell maturation antigen
  • CD269 and TNFRSF17 are tumor necrosis family receptor (TNFR) members preferentially expressed in differentiated plasma cells, and involved in mediating the survival of the plasma cells for maintaining long-term humoral immunity.
  • TNFR tumor necrosis family receptor
  • BCMA is a non-glycosylated type I transmembrane protein, which is involved in B cell maturation, growth and survival.
  • BCMA is a receptor for two ligands of the TNF superfamily: APRIL (a proliferation-inducing ligand, CD256, TNFSF13) , the high-affinity ligand to BCMA and the B cell activation factor BAFF (THANK, BlyS, B lymphocyte stimulator, TALL-1 and zTNF4) , the low-affinity ligand to BCMA.
  • APRIL and BAFF show structural similarity and overlapping yet distinct receptor binding specificity.
  • the negative regulator TACI also binds to both BAFF and APRIL.
  • the coordinate binding of APRIL and BAFF to BCMA and/or TACI activates transcription factor NF- ⁇ and increases the expression of pro-survival Bcl-2 family members (e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, Al) and down-regulates expression of pro-apoptotic factors (e.g., Bid, Bad, Bik, Bim, etc. ) , thus inhibiting apoptosis and promoting survival.
  • pro-survival Bcl-2 family members e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, Al
  • pro-apoptotic factors e.g., Bid, Bad, Bik, Bim, etc.
  • BCMA also supports growth and survival of malignant human B cells.
  • the expression of BCMA has been linked to a number of cancers, autoimmune disorders, and infectious diseases. Cancers with increased expression of BCMA include some hematological cancers, such as multiple myeloma, Hodgkin’s and non-Hodgkin’s lymphoma, various leukemias, and glioblastoma.
  • Anti-BCMA single domain antibodies have been identified herein. The instantly disclosed ones exhibited high specificity and superior affinity. Some antibodies also exhibited cross-reactivity to both human and cynomolgus BCMA proteins. Meanwhile, given that these single domain antibodies are single-chain and small in size, they are better suited for use in multifunctional antibodies including T cell engagers, NK cell engagers, multifunctional proteins, bispecific or trispecific antibodies and chimeric antigen receptor (CAR) -related therapies.
  • CAR chimeric antigen receptor
  • a single domain antibody or a polypeptide comprising the single domain antibody, wherein the single domain antibody has binding specificity to the B-cell maturation antigen (BCMA) protein and comprises the CDR1, CDR2 and CDR3 of any one of the antibodies A016, A022, A026, A034, A043, A052, A058, A060, A068, A075, A077, A081, A095, A098, A119, A130, A134, A135, A137, B010, B030, B077, B081, B109, B127, B133, B136, B168, B171, B172, B192, B231, B255, B270, B281, B282 and B208.
  • the sequences of these antibodies are provided in Tables 1 and 3 and example CDR sequences (e.g., according to Kabat numbering) are shown in Tables 2 and 4.
  • the CDR1, CDR2, and CDR3, respectively comprise the amino acid sequences of: SEQ ID NO: 20, 21 and 22; SEQ ID NO: 23, 24 and 22; SEQ ID NO: 25, 26 and 27; SEQ ID NO: 28, 29 and 22; SEQ ID NO: 30, 31 and 32; SEQ ID NO: 33, 34 and 35;SEQ ID NO: 36, 37 and 38; SEQ ID NO: 39, 40 and 41; SEQ ID NO: 42, 43 and 44; SEQ ID NO: 45, 46 and 47; SEQ ID NO: 48, 49 and 50; SEQ ID NO: 51, 52 and 53; SEQ ID NO: 54, 55 and 56; SEQ ID NO: 57, 58 and 59; SEQ ID NO: 60, 61 and 62; SEQ ID NO: 63, 64 and 65; SEQ ID NO: 66, 67 and 68; SEQ ID NO: 69, 70 and 71; or SEQ ID NO: 72, 73 and 74.
  • the CDR1, CDR2, and CDR3, respectively comprise the amino acid sequences of: SEQ ID NO: 92, 93 and 94; SEQ ID NO: 95, 96 and 97; SEQ ID NO: 92, 98 and 99; SEQ ID NO: 100, 101 and 102; SEQ ID NO: 103, 104 and 94; SEQ ID NO: 105, 106 and 107; SEQ ID NO: 105, 108 and 109; SEQ ID NO: 92, 110 and 99; SEQ ID NO: 92, 111 and 99; SEQ ID NO: 103, 111 and 112; SEQ ID NO: 95, 96 and 113; SEQ ID NO: 103, 114 and 99; SEQ ID NO: 92, 115 and 116; SEQ ID NO: 103, 117 and 116; SEQ ID NO: 92, 118 and 116; SEQ ID NO: 92, 119 and 112; SEQ ID NO: 103, 124 and 112;
  • the antibody or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-19, or amino acid sequence having at least 80%sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1-19 while retaining the CDR of the corresponding sequence. In some embodiments, antibody or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-19.
  • Chimeric antigen receptors CAR
  • bispecific antibodies bispecific antibodies
  • trispecific antibodies multifunctional proteins
  • multifunctional proteins multifunctional proteins
  • cells engineered to express any of these proteins are also provided, in various embodiments.
  • method of treating cancer in a patient in need thereof comprising administering to the patient the antibody or polypeptide, the CAR, a polynucleotide encoding them, a cell engineered to express each, or an antibody-drug conjugate of the present disclosure.
  • the cancer is a hematological cancer, such as a BCMA-expressing B cell cancer (e.g., multiple myeloma) .
  • FIG. 1 shows the binding of the candidate antibodies from single-domain library to BCMA-HEK293, HEK293 and CHO-S cells as measured by FACS.
  • FIG. 2 shows the result of the non-specific binding test of the candidate antibodies from single-domain library to both HEK293 cells as measured by FACS.
  • FIG. 3 shows the result of the non-specific binding test of the candidate antibodies from single-domain library to both CHO-S cells as measured by FACS.
  • FIG. 4 shows the binding of the candidate antibodies from single-domain library to MM. 1S cells as measured by FACS.
  • FIG. 5 shows the FACS binding of the candidate antibodies from single-domain library to NCI-H929 cells.
  • FIG. 6 shows the FACS binding of the candidate antibodies from single-domain library to U266 cells.
  • FIG. 7 shows the FACS binding of the candidate antibodies from single-domain library to OPM-2 cells.
  • FIG. 8 shows the FACS binding of candidate antibodies from alpaca immune library to NCI-H929 cells.
  • FIG. 9 shows FACS binding of candidate antibodies from alpaca immune library to U266 cells.
  • FIG. 10 shows FACS binding of candidate antibodies from alpaca immune library to CHO-S cells.
  • FIG. 11 shows FACS binding of candidate antibodies from alpaca immune library to HEK293 cells.
  • FIG. 12 shows FACS binding of candidate antibodies from alpaca immune library to Jurkat cells.
  • a or “an” entity refers to one or more of that entity; for example, “an antibody, ” is understood to represent one or more antibodies.
  • the terms “a” (or “an” ) , “one or more, ” and “at least one” can be used interchangeably herein.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 98 %or 99 %) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment.
  • One alignment program is BLAST, using default parameters.
  • Biologically equivalent polynucleotides are those having the above-noted specified percent homology and encoding a polypeptide having the same or similar biological activity.
  • an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen.
  • An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof.
  • the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen.
  • CDR complementarity determining region
  • antibody fragment or “antigen-binding fragment” , as used herein, is a portion of an antibody such as F (ab’ ) 2 , F (ab) 2 , Fab’ , Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody.
  • antibody fragment includes aptamers, spiegelmers, and diabodies.
  • antibody fragment also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • a “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (V H ) and light chains (V L ) of immunoglobulins.
  • the regions are connected with a short linker peptide of ten to about 25 amino acids.
  • the linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V H with the C-terminus of the V L , or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.
  • ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.
  • antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g., ⁇ l- ⁇ 4) . It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.
  • the immunoglobulin subclasses isotypes) e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgG 5 , etc.
  • immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.
  • IgG a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000.
  • the four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
  • Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab’ and F (ab’ ) 2 , Fd, Fvs, single-chain Fvs (scFv) , single-chain antibodies, disulfide-linked Fvs (sdFv) , fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies disclosed herein) .
  • anti-Id antigen-binding polypeptides, variants, or derivatives thereof of the disclosure
  • Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • type e.g., IgG, IgE, IgM, IgD, IgA, and IgY
  • class e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2
  • subclass of immunoglobulin molecule e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2
  • a single-domain antibody also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain.
  • Nanobodies produced from camelids, alpacas and certain other animals are also referred to as VHH fragments. Like a whole antibody, a nanobody is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, single domain antibodies are much smaller than common antibodies (150–160 kDa) .
  • an antibody By “specifically binds” or “has specificity to, ” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.
  • the term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope.
  • antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B, ” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D. ”
  • the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable.
  • “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • subject or “individual” or “animal” or “patient” or “mammal, ” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
  • phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.
  • CDR complementarity determining region
  • Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody.
  • One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself.
  • “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983) .
  • top antibodies included B010, B030, B077, B081, B109, B127, B133, B136, B168, B171, B172, B192, B231, B255, B270, B281, B282 and B208, which are also considered suitable for further clinical development.
  • single domain antibodies having binding specificity to a human B-cell maturation antigen (BCMA) protein.
  • BCMA B-cell maturation antigen
  • single domain antibodies and polypeptides that include such a single domain antibody.
  • the single domain antibody includes a CDR1, a CDR2 and a CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of any of the identified antibodies A016, A022, A026, A034, A043, A052, A058, A060, A068, A075, A077, A081, A095, A098, A119, A130, A134, A135 and A137.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A016 (SEQ ID NO: 1) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 20
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 21
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 22.
  • the antibody includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 1. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A016. Also provided, in some embodiments, are antibodies that compete with A016 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A022 (SEQ ID NO: 2) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 23
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 24
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 22.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A026 (SEQ ID NO: 3) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 25
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 26
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 27.
  • the antibody includes the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 3. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A026. Also provided, in some embodiments, are antibodies that compete with A034 in binding to BCMA.
  • the antibody includes the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 4. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A034. Also provided, in some embodiments, are antibodies that compete with A034 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A043 (SEQ ID NO: 5) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 30
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 31
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 32.
  • the antibody includes the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 5. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A043. Also provided, in some embodiments, are antibodies that compete with A043 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A058 (SEQ ID NO: 7) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 36
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 37
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 38.
  • the antibody includes the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 7. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A058. Also provided, in some embodiments, are antibodies that compete with A058 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A060 (SEQ ID NO: 8) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 39
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 40
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 41.
  • the antibody includes the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody includes the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 8. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A060. Also provided, in some embodiments, are antibodies that compete with A060 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A068 (SEQ ID NO: 9) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 42
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 43
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 44.
  • the antibody includes the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 9. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A068. Also provided, in some embodiments, are antibodies that compete with A068 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A075 (SEQ ID NO: 10) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 45
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 46
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 47.
  • the antibody includes the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 10. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A075. Also provided, in some embodiments, are antibodies that compete with A075 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A077 (SEQ ID NO: 11) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 48
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 49
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 50.
  • the antibody includes the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 11. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A077. Also provided, in some embodiments, are antibodies that compete with A077 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A081 (SEQ ID NO: 12) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 51
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 52
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 53.
  • the antibody includes the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 12. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A081. Also provided, in some embodiments, are antibodies that compete with A081 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A095 (SEQ ID NO: 13) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 54
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 55
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 56.
  • the antibody includes the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 13. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A095. Also provided, in some embodiments, are antibodies that compete with A095 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A024 (SEQ ID NO: 14) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 57
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 58
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 59.
  • the antibody includes the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody includes the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 14. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A024. Also provided, in some embodiments, are antibodies that compete with A024 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A119 (SEQ ID NO: 15) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 60
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 61
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 62.
  • the antibody includes the amino acid sequence of SEQ ID NO: 15. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 15. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A119. Also provided, in some embodiments, are antibodies that compete with A119 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A130 (SEQ ID NO: 16) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 63
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 64
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 65.
  • the antibody includes the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 16. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A130. Also provided, in some embodiments, are antibodies that compete with A130 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A134 (SEQ ID NO: 17) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 66
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 67
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 68.
  • the antibody includes the amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 17. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A134. Also provided, in some embodiments, are antibodies that compete with A134 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A135 (SEQ ID NO: 18) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 69
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 70
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 71.
  • the antibody includes the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 18. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A135. Also provided, in some embodiments, are antibodies that compete with A135 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A137 (SEQ ID NO: 19) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 72
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 73
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 74.
  • the antibody includes the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 19. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A137. Also provided, in some embodiments, are antibodies that compete with A137 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B010 (SEQ ID NO: 75) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 92
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 93
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 94.
  • the antibody includes the amino acid sequence of SEQ ID NO: 75. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 75. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B010. Also provided, in some embodiments, are antibodies that compete with B010 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B030 (SEQ ID NO: 76) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 95
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 96
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 97.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B077 (SEQ ID NO: 77) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 92
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 98
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 99.
  • the antibody includes the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 77. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B077. Also provided, in some embodiments, are antibodies that compete with B077 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B081 (SEQ ID NO: 78) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 100
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 101
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 102.
  • the antibody includes the amino acid sequence of SEQ ID NO: 78. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 78. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B081. Also provided, in some embodiments, are antibodies that compete with B081 in binding to BCMA.
  • the antibody includes the amino acid sequence of SEQ ID NO: 79. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 79. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B109. Also provided, in some embodiments, are antibodies that compete with B109 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B127 (SEQ ID NO: 80) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 105
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 106
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 107.
  • the antibody includes the amino acid sequence of SEQ ID NO: 80. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 80. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B127. Also provided, in some embodiments, are antibodies that compete with B127 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B133 (SEQ ID NO: 81) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 105
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 108
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 109.
  • the antibody includes the amino acid sequence of SEQ ID NO: 82. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 82. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B136. Also provided, in some embodiments, are antibodies that compete with B136 in binding to BCMA.
  • the antibody includes the amino acid sequence of SEQ ID NO: 83. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 83. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B168. Also provided, in some embodiments, are antibodies that compete with B168 in binding to BCMA.
  • the antibody includes the amino acid sequence of SEQ ID NO: 84. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 84. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B171. Also provided, in some embodiments, are antibodies that compete with B171 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B172 (SEQ ID NO: 85) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 95
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 96
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 113.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B192 (SEQ ID NO: 86) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 103
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 114
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 99.
  • the antibody includes the amino acid sequence of SEQ ID NO: 86. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 86. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B192. Also provided, in some embodiments, are antibodies that compete with B192 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B231 (SEQ ID NO: 87) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 92
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 115
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 116.
  • the antibody includes the amino acid sequence of SEQ ID NO: 87. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 87. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B231. Also provided, in some embodiments, are antibodies that compete with B231 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B255 (SEQ ID NO: 88) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 103
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 117
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 116.
  • the antibody includes the amino acid sequence of SEQ ID NO: 88. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 88. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B255. Also provided, in some embodiments, are antibodies that compete with B255 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B270 (SEQ ID NO: 89) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 92
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 118
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 116.
  • the antibody includes the amino acid sequence of SEQ ID NO: 89. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 89. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B270. Also provided, in some embodiments, are antibodies that compete with B270 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B281 (SEQ ID NO: 90) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 92
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 119
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 112.
  • the antibody includes the amino acid sequence of SEQ ID NO: 90. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 90. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B281. Also provided, in some embodiments, are antibodies that compete with B281 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B282 (SEQ ID NO: 123) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 103
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 124
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 112.
  • the antibody includes the amino acid sequence of SEQ ID NO: 123. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 123. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B282. Also provided, in some embodiments, are antibodies that compete with B282 in binding to BCMA.
  • the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B208 (SEQ ID NO: 91) .
  • the CDR1 includes the amino acid sequence of SEQ ID NO: 120
  • the CDR2 includes the amino acid sequence of SEQ ID NO: 121
  • the CDR3 includes the amino acid sequence of SEQ ID NO: 122.
  • the antibody includes the amino acid sequence of SEQ ID NO: 91. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 91. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B208. Also provided, in some embodiments, are antibodies that compete with B208 in binding to BCMA.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived.
  • a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%identical to the starting sequence.
  • Antibodies, variants, or derivatives thereof of the disclosure include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to the epitope.
  • the antibodies can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • the antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
  • the antibodies of the disclosure are covalently attached to a drug moiety.
  • the drug moiety may be, or be modified to include, a group reactive with a conjugation point on the antibody.
  • a drug moiety can be attached by alkylation (e.g., at the epsilon-amino group lysines or the N-terminus of antibodies) , reductive amination of oxidized carbohydrate, transesterification between hydroxyl and carboxyl groups, amidation at amino groups or carboxyl groups, and conjugation to thiols.
  • the number of drug moieties, p, conjugated per antibody molecule ranges from an average of 1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from an average of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In other embodiments, p is an average of 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, p ranges from an average of about 1 to about 20, about 1 to about 10, about 2 to about 10, about 2 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2. In some embodiments, p ranges from about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4 or about 2 to about 3.
  • the protein when chemical activation of the protein results in formation of free thiol groups, the protein may be conjugated with a sulfhydryl reactive agent.
  • the agent is one which is substantially specific for free thiol groups.
  • agents include, for example, malemide, haloacetamides (e.g., iodo, bromo or chloro) , haloesters (e.g., iodo, bromo or chloro) , halomethyl ketones (e.g., iodo, bromo or chloro) , benzylic halides (e.g., iodide, bromide or chloride) , vinyl sulfone and pyridylthio.
  • haloacetamides e.g., iodo, bromo or chloro
  • haloesters e.g., iodo, bromo or chloro
  • halomethyl ketones
  • the drug can be linked to the antibody or fragment by a linker.
  • Suitable linkers include, for example, cleavable and non-cleavable linkers.
  • a cleavable linker is typically susceptible to cleavage under intracellular conditions.
  • Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease.
  • Sulfo-smcc conjugation occurs via a maleimide group which reacts with sulfhydryls (thiols, -SH) , while its Sulfo-NHS ester is reactive toward primary amines (as found in Lysine and the protein or peptide N-terminus) .
  • Another linker is maleimidocaproyl (mc) .
  • suitable linkers include linkers hydrolyzable at a specific pH or a pH range, such as a hydrazone linker.
  • Additional suitable cleavable linkers include disulfide linkers. The linker may be covalently bound to the antibody to such an extent that the antibody must be degraded intracellularly in order for the drug to be released e.g. the mc linker and the like.
  • a linker can include a group for linkage to the antibody.
  • linker can include an amino, hydroxyl, carboxyl or sulfhydryl reactive groups (e.g., malemide, haloacetamides (e.g., iodo, bromo or chloro) , haloesters (e.g., iodo, bromo or chloro) , halomethyl ketones (e.g., iodo, bromo or chloro) , benzylic halides (e.g., iodide, bromide or chloride) , vinyl sulfone and pyridylthio) .
  • amino, hydroxyl, carboxyl or sulfhydryl reactive groups e.g., malemide, haloacetamides (e.g., iodo, bromo or chloro) , haloesters (e.g., iodo,
  • the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressive agent, a radioisotope, a toxin, or the like.
  • the conjugate can be used for inhibiting the multiplication of a tumor cell or cancer cell, causing apoptosis in a tumor or cancer cell, or for treating cancer in a patient.
  • the conjugate can be used accordingly in a variety of settings for the treatment of animal cancers.
  • the conjugate can be used to deliver a drug to a tumor cell or cancer cell.
  • the conjugate binds to or associates with a cancer cell expressing BCMA, and the conjugate and/or drug can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis.
  • Examples of drug moieties or payloads are selected from the group consisting of DM1 (maytansine, N2’ -deacetyl-N2’ - (3-mercapto-1-oxopropyl) -or N2’ -deacetyl-N2’ - (3-mercapto-1-oxopropyl) -maytansine) , mc-MMAD (6-maleimidocaproyl-monomethylauristatin-D or N-methyl-L-valyl-N- [ (1S, 2R) -2-methoxy-4- [ (2S) -2- [ (1R, 2R) -1-methoxy-2-methyl-3-oxo-3- [ [ (1S) -2-phenyl-1- (2-thiazolyl) ethyl] amino] propyl] -1-pyr rolidinyl] -1- [ (1S) -1-methylpropyl] -4-oxobutyl] -N-
  • DM1 is a derivative of the tubulin inhibitor maytansine while MMAD, MMAE, and MMAF are auristatin derivatives.
  • the drug moiety is selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE.
  • the drug moiety is a maytansinoid or an auristatin.
  • the antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.
  • a therapeutic agent which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.
  • the antibodies can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • the antibodies can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) .
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the second antigen is a protein expressed on an immune cell, such as a T cell, a B cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a phagocyte, a natural killer cell, an eosinophil, a basophil, and a mast cell.
  • an immune cell such as a T cell, a B cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a phagocyte, a natural killer cell, an eosinophil, a basophil, and a mast cell.
  • the second antigen is GPRC5D, CD123, CS1, CD20, CD19, CD22, CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.
  • Bifunctional molecules that include not just antibody or antigen binding fragment are also provided.
  • an antibody or antigen-binding fragment specific to BCMA such as those described here, can be combined with an immune cytokine or ligand optionally through a peptide linker.
  • the linked immune cytokines or ligands include, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF- ⁇ , CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL.
  • Such bi-functional molecules can combine the immune checkpoint blocking effect with tumor site local immune modulation.
  • the present disclosure provides a T-cell engager that includes an antibody or polypeptide of the present disclosure.
  • T-cell engagers can come with different molecular designs and can be particularly useful for the treatment of liquid or solid tumors.
  • the T cell engager is bispecific, which can target BCMA and GPRC5D.
  • the T cell engager is trispecific, which can target BCMA and GPRC5D, and yet another epitope on BCMA or another tumor antigen.
  • the transmembrane domain can include a sequence that spans a cell membrane, but extends into the cytoplasm of a cell and/or into the extracellular space.
  • a transmembrane can include a membrane-spanning sequence which itself can further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm of a cell, and/or the extracellular space.
  • a transmembrane domain includes a membrane-spanning region, yet can further comprise an amino acid (s) that extend beyond the internal or external surface of the membrane itself; such sequences can still be considered to be a “transmembrane domain” .
  • the transmembrane domain is fused to the cytoplasmic domain through a short linker.
  • the short peptide or polypeptide linker preferably between 2 and 10 amino acids in length can form the linkage between the transmembrane domain and a proximal cytoplasmic signaling domain of the chimeric receptor.
  • a glycine-serine doublet (GS) , glycine-serine-glycine triplet (GSG) , or alanine-alanine-alanine triplet (AAA) provides a suitable linker.
  • the CAR further includes a costimulatory domain.
  • the costimulatory domain is positioned between the transmembrane domain and an activating domain.
  • Example costimulatory domains include, but are not limited to, CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8, CD11a (ITGAL) , CD11b (ITGAM) , CD11c (ITGAX) , CD11d (ITGAD) , CD18 (ITGB2) , CD19 (B4) , CD27 (T FRSF7) , CD28, CD28T, CD29 (ITGB1) , CD30 (TNFRSF8) , CD40 (TNFRSF5) , CD48 (SLAMF2) , CD49a (ITGA1) , CD49d (ITGA4) , CD49f (ITGA6) , CD66a (CEACAM1) , CD66b (CEACAM8) , CD66c (CEACAM
  • the cytoplasmic portion of the CAR also includes a signaling/activation domain.
  • the signaling/activation domain is the CD3 ⁇ domain, or is an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98%or 99%sequence identity to the CD3 ⁇ domain.
  • Bispecific and bicistronic CARs are also provided.
  • the CAR protein further includes a second antigen-binding fragment (e.g., scFv) that binds a second target.
  • a second antigen-binding fragment e.g., scFv
  • Non-limiting examples of the second target include GPRC5D, CD123, CS1, CD20, CD19, CD22, CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, and VEGF.
  • a bicistronic CAR includes two CAR molecules that may be expressed by the same vector encoding both. Both CAR molecules can be expressed in a target cell, such as a T cell.
  • Cells that enclose the CAR or the encoding nucleotides are also provided in the present disclosure.
  • a suitable cell can be used, that is transduced with a vector that encodes, or put in contact with, an CAR that includes an anti-BCMA antibody of the present disclosure (or alternatively engineered to express an anti-BCMA antibody of the present disclosure) .
  • the cell e.g., T cell, NK cell, monocyte, macrophage
  • T cell e.g., T cell, NK cell, monocyte, macrophage
  • the cell can be, for instance, a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, a gamma delta T cell, or the combination thereof, without limitation.
  • Polynucleotides, mRNA, and Methods of Expressing or Preparing Antibodies are also provided in the present disclosure.
  • the present disclosure also provides polynucleotides or nucleic acid molecules encoding the antibodies, variants or derivatives thereof of the disclosure, or the CAR.
  • the polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.
  • the polynucleotide is an mRNA molecule.
  • the mRNA can be introduced into a target cell for expressing the antibody or fragment thereof.
  • mRNAs may be synthesized according to any of a variety of known methods.
  • the mRNAs may be synthesized via in vitro transcription (IVT) .
  • IVT in vitro transcription
  • a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase) , DNAse I, pyrophosphatase, and/or RNAse inhibitor.
  • RNA polymerase e.g., T3, T7 or SP6 RNA polymerase
  • a DNA template is transcribed in vitro.
  • a suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired antibody encoding (e.g., heavy chain or light chain encoding) mRNA and a termination signal.
  • Desired antibody encoding (e.g., heavy chain or light chain encoding) mRNA sequence may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., a desired heavy chain or light chain sequence) , a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.
  • a desired amino acid sequence e.g., a desired heavy chain or light chain sequence
  • optimization algorithms may then be used for selection of suitable codons.
  • the mRNA may be synthesized as unmodified or modified mRNA.
  • mRNAs are modified to enhance stability.
  • Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA.
  • a modified mRNA can thus include, for example, backbone modifications, sugar modifications or base modifications.
  • antibody encoding mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A) , guanine (G) ) or pyrimidines (thymine (T) , cytosine (C) , uracil (U) ) , and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g.
  • the mRNAs may contain RNA backbone modifications.
  • a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically.
  • Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5’ -O- (1-thiophosphate) ) , boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.
  • the mRNAs may contain sugar modifications.
  • a typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2’ -deoxy-2’ -fluoro-oligoribonucleotide (2’ -fluoro-2’ -deoxycytidine 5’ -triphosphate, 2’ -fluoro-2’ -deoxyuridine 5’ -triphosphate) , 2’ -deoxy-2’ -deamine-oligoribonucleotide (2’ -amino-2’ -deoxycytidine 5’ -triphosphate, 2’ -amino-2’ -deoxyuridine 5’ -triphosphate) , 2’ -O-alkyloligoribonucleotide, 2’ -deoxy-2’ -C-alkyloligoribonucleotide
  • the mRNAs may contain modifications of the bases of the nucleotides (base modifications) .
  • a modified nucleotide which contains a base modification is also called a base-modified nucleotide.
  • base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine riboside 5’ -triphosphate, 2-aminoadenosine 5’ -triphosphate, 2-thiocytidine 5’ -triphosphate, 2-thiouridine 5’ -triphosphate, 4-thiouridine 5’ -triphosphate, 5-aminoallylcytidine 5’ -triphosphate, 5-aminoallyluridine 5’ -triphosphate, 5-bromocytidine 5’ -triphosphate, 5-bromouridine 5’ -triphosphate, 5-iodocytidine 5’ -triphosphate, 5-iodouridine 5’ -triphosphate, 5-methylcytidine 5’ -triphosphate, 5-methyluridine 5’ -triphosphate, 6-azacytidine 5’ -triphosphate, 6-azauridine 5’ -triphosphate, 6-chloropurine riboside 5’
  • mRNA synthesis includes the addition of a “cap” on the N-terminal (5’ ) end, and a “tail” on the C-terminal (3’ ) end.
  • the presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells.
  • the presence of a “tail” serves to protect the mRNA from exonuclease degradation.
  • the mRNAs include a 5’ cap structure.
  • a 5’ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5’ 5’ 5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
  • GTP guanosine triphosphate
  • cap structures include, but are not limited to, m7G (5’ ) ppp (5’ (A, G (5’ ) ppp (5) A and G (5) ppp (5’ ) G.
  • the mRNAs include a 3’ poly (A) tail structure.
  • a poly-A tail on the 3’ terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 125 adenosine nucleotides, 10 to 100 adenosine nucleotides, about 10 to 75 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides) .
  • antibody encoding mRNAs include a 3’ poly (C) tail structure.
  • a suitable poly-C tail on the 3’ terminus of mRNA typically includes about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides) .
  • the poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.
  • the mRNAs include a 5’ and/or 3’ untranslated region.
  • a 5’ untranslated region includes one or more elements that affect an mRNA’s stability or translation, for example, an iron responsive element.
  • a 5’ untranslated region may be between about 50 and 500 nucleotides in length (e.g., about 50 and 400 nucleotides in length, about 50 and 300 nucleotides in length, about 50 and 200 nucleotides in length, or about 50 and 100 nucleotides in length) .
  • the present technology may be used to deliver any antibody known in the art and antibodies that can be produced against desired antigens using standard methods.
  • the present invention may be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, or bi-specific antibodies.
  • both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human.
  • Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. patents: 6,150,584; 6,458,592; 6,420,140 which are incorporated by reference in their entireties. Treatment and Uses
  • the antibodies, variants, antibody-drug conjugates, chimeric antigen receptors (CAR) and CAR cells, encoding polynucleotides or derivatives of the present disclosure may be used in certain treatment and diagnostic methods.
  • the present disclosure is further directed to antibody-based therapies which involve administering the antibodies or fragments, antibody-drug conjugates, chimeric antigen receptors (CAR) and CAR cells, encoding polynucleotides or derivatives of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein.
  • Therapeutic molecules or cells of the disclosure include, but are not limited to, antibodies of the disclosure (including variants and derivatives thereof as described herein) , antibody-drug conjugates, chimeric antigen receptors (CAR) and CAR cells, and nucleic acids or polynucleotides encoding antibodies of the disclosure (including variants and derivatives thereof as described herein) .
  • the method in one embodiment, entails administering to the patient an effective amount of the molecules or cells of the present disclosure.
  • at least one of the cancer cells (e.g., stromal cells) in the patient over-express BCMA.
  • Cellular therapies such as chimeric antigen receptor (CAR) T-cell therapies, are also provided in the present disclosure.
  • a suitable cell can be used, that is transduced with a vector that encodes, or put in contact with, a CAR that includes an anti-BCMA antibody of the present disclosure (or alternatively engineered to express an anti-BCMA antibody of the present disclosure) .
  • the cell can then be introduced to a cancer patient in need of treatment.
  • the cancer patient may have a cancer of any of the types as disclosed herein.
  • the cell e.g., T cell, NK cell, monocyte, macrophage
  • T cell can be, for instance, a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, a gamma delta T, or the combination thereof, without limitation.
  • the cell was isolated from the cancer patient him-or her-self. In some embodiments, the cell was provided by a donor or from a cell bank. When the cell is isolated from the cancer patient, undesired immune reactions can be minimized.
  • Non-limiting examples of cancers include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.
  • the cancer is one or more of gastric, pancreatic, esophageal, ovarian, and lung cancers.
  • Additional diseases or conditions associated with increased cell survival include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) ) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia) ) , polycythemia vera, lymphomas (e.g., Hodgkin’s disease and non-Hodgkin’s disease) , multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sar
  • MM-affected patients may experience a variety of disease-related symptoms due to, bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and the psychosocial burden of a cancer diagnosis.
  • the 5-year relative survival rate for MM was approximately 34%highlighting that MM is a difficult-to-treat disease where there are currently no curative options.
  • Methods of administration of the antibody or fragment include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the antigen-binding polypeptides or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc. ) and may be administered together with other biologically active agents.
  • compositions containing the antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch) , buccally, or as an oral or nasal spray.
  • Administration can be systemic or local.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • the amount of the antibodies of the disclosure which will be effective in the treatment, inhibition and prevention of an inflammatory, immune or malignant disease, disorder or condition can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, disorder or condition, and should be decided according to the judgment of the practitioner and each patient’s circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the dosage administered to a patient of the antibodies of the present disclosure is typically 0.001 mg/kg to 100 mg/kg of the patient’s body weight, between 0.01 mg/kg and 20 mg/kg of the patient’s body weight, or 0.5 mg/kg to 10 mg/kg of the patient’s body weight.
  • human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
  • the dosage and frequency of administration of antibodies of the disclosure may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
  • compositions of the disclosure are administered in combination with cytokines.
  • Cytokines that may be administered with the compositions of the disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF- ⁇ .
  • compositions of the disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
  • compositions of the disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
  • compositions comprise an effective amount of an antibody, antibody-drug conjugates, chimeric antigen receptors (CAR) and CAR cells, encoding polynucleotides, or derivatives and an acceptable carrier.
  • the composition further includes a second anticancer agent (e.g., an immune checkpoint inhibitor) .
  • the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the compounds of the disclosure can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • This example demonstrates the screening for positive clones through panning of a synthetic, humanized single domain library and primary screening, where Hu-BCMA-ECD-His, Hu-BCMA-ECD-Fc, Cyno-BCMA-ECD-His, Cyno-BCMA-ECD-Fc antigens and Hu-BCMA-HEK293, NCI-H929 were used as antigens (or controls) .
  • Dynabeads blocking fresh prepared magnetic beads were blocked with 2.5%BSA.
  • coated beads were incubated with depleted phages, and further washed according to the Liquid-phase magnetic bead screening system method.
  • Infection and cultivation collected phages were mixed with the logarithmic phase SS320 cells and incubated for 30 min at 37°C. After the incubation, spreading of infected cells on the 2YT-Car+-Tet+ plate was performed and incubation was conducted at 37°C overnight in an incubator.
  • Immunotube coating and blocking the diluted antigens were added into the immunotubes and coat overnight at 4°C, then washed and blocked with 5%PBSM.
  • phage suspensions were incubated in antigen-coated immunotubes. Binding and subsequent wash processes were conducted according to the immunotube screening system method.
  • Infection and cultivation collected phages were mixed with the logarithmic phase SS320 cells and incubated for 30 min at 37°C. After the incubation, spreading of infected cells on the 2YT-Car+-Tet+ plate was performed and incubation was conducted at 37°C overnight in an incubator.
  • Phage blocking the prepared phage libraries were blocked with 5%PBSM at room temperature on the four-dimensional rotary instrument for 1 h. Incubation
  • Negative panning blocked phage suspensions were added into the negative cell solution, and then the mixture was placed on orbital shaker at (room temperature, 80 rpm, 1 hour) . Notice: phage suspension was added carefully in order not to wash out the cell.
  • Positive panning negatively screened phages were added into the corresponding positive cell bottles using a disposable sucker and the mixture was incubated on orbital shaker (room temperature, 80 rpm, 2 hours) .
  • Infection and cultivation collected phages were mixed with the logarithmic phase SS320 cells and incubated for 30 min at 37°C. After the incubation, spreading of infected cells on the 2YT-Car+-Tet+ plate was performed and incubation was conducted at 37°C overnight in an incubator.
  • Blocking Block with 5%PBSM at room temperature for 1 h, wash the plates 3 times with PBST.
  • Secondary antibody Add 1: 5000 diluted secondary antibody Anti-Flag-HRP to VHH supernatant, dilute with PBS, 30 ⁇ L/well, place at room temperature for 1 h, wash the plate 6 times with PBST; Add 1: 20000 diluted secondary antibody Anti-M13-HRP to Phage, dilute with 5%PBSM, 30 ⁇ L/well, place at room temperature for 1 h, then wash the plate 6 times with PBST.
  • Blocking Block with 5%PBSM at room temperature for 1 h, wash the plates 3 times with PBST.
  • Secondary antibody Add 30 ⁇ L of diluted secondary antibody Anti-VHH-HRP (1: 7000) , place at room temperature for 1 h, then wash the plate 6 times with PBST.
  • Cells were processed into cell suspension, and the density was adjusted to 1 ⁇ 10 6 cells/mL.
  • the cell suspension was added to a 96-well round-bottom plate at 100 ⁇ L per well using a 100 ⁇ L pipette, followed by centrifugation at 300 g for 5 min to discard the supernatant. Addition of antibody diluent
  • Cells were processed into cell suspension, and the density was adjusted to 1 ⁇ 10 6 cells/mL.
  • the cell suspension was added to a 96-well round-bottom plate at 100 ⁇ L per well using a 100 ⁇ L pipette, followed by centrifugation at 300 g for 5 min to discard the supernatant. Addition of antibody diluent
  • the antibody was diluted with FACS Buffer to 8 gradient concentrations of 300.0000, 75.0000, 18.7500, 4.6875, 1.1719, 0.2930, 0.0732, and 0.0183 nM for NCI-H929 and U266 cells binding test.
  • Antibodies to HEK293, CHO-S, and Jurkat cells were diluted with FACS Buffer into dilutions of 2 gradient concentrations of 300.0000 and 100.0000 nM.
  • the antibody diluent was added to a plate at 100 ⁇ L per well using a 100 ⁇ L pipette, and incubated at 4°C for 60 min. Plate washing: The cells were washed twice with the FACS Buffer. Addition of secondary antibody
  • the candidates exhibited binding to NCI-H929 and U266 tumor cells.
  • 8 candidate antibodies i.e., B010, B030, B133, B136, B168, B172, B077, and B192, exhibited potent binding to both NCI-H929 and U266 cells. None of the 8 candidate antibodies exhibited non-specific binding to HEK293, CHO-S, or Jurkat cells.

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Abstract

Provided are single domain antibodies having binding specificity to the human BCMA protein. Such antibodies, as well as their multi-specific counterparts and chimeric antigen receptors are capable of targeting cancer cells expressing BCMA, and thus can be used to treat the cancer, in particular hematological cancers.

Description

SINGLE DOMAIN ANTIBODIES FOR BCMA
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of International Application No. PCT/CN2023/140105, filed December 20, 2023, the content of which is hereby incorporated by reference in its entirety.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
The content of the electronic sequence listing (371414. xml; Size: 112, 790 bytes; and Date of Creation: December 13, 2024) is herein incorporated by reference in its entirety.
BACKGROUND
B-cell maturation antigen (BCMA) , also known as CD269 and TNFRSF17 (UniProt Q02223) , is a tumor necrosis family receptor (TNFR) member preferentially expressed in differentiated plasma cells, and involved in mediating the survival of the plasma cells for maintaining long-term humoral immunity. BCMA is a non-glycosylated type I transmembrane protein, which is involved in B cell maturation, growth and survival.
BCMA is a receptor for two ligands of the TNF superfamily: APRIL (a proliferation-inducing ligand, CD256, TNFSF13) , the high-affinity ligand to BCMA and the B cell activation factor BAFF (THANK, BlyS, B lymphocyte stimulator, TALL-1 and zTNF4) , the low-affinity ligand to BCMA. APRIL and BAFF show structural similarity and overlapping yet distinct receptor binding specificity. The negative regulator TACI also binds to both BAFF and APRIL. The coordinate binding of APRIL and BAFF to BCMA and/or TACI activates transcription factor NF-κΒ and increases the expression of pro-survival Bcl-2 family members (e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, Al) and down-regulates expression of pro-apoptotic factors (e.g., Bid, Bad, Bik, Bim, etc. ) , thus inhibiting apoptosis and promoting survival. This combined action promotes B cell differentiation, proliferation, survival and antibody production.
In line with the above finding, BCMA also supports growth and survival of malignant human B cells. The expression of BCMA has been linked to a number of cancers, autoimmune disorders, and infectious diseases. Cancers with increased expression of BCMA include some hematological cancers, such as multiple myeloma, Hodgkin’s and non-Hodgkin’s lymphoma, various leukemias, and glioblastoma.
SUMMARY
Anti-BCMA single domain antibodies have been identified herein. The instantly disclosed ones exhibited high specificity and superior affinity. Some antibodies also exhibited cross-reactivity to both human and cynomolgus BCMA proteins. Meanwhile, given that these single domain antibodies are single-chain and small in size, they are better suited for use in multifunctional antibodies including T cell engagers, NK cell engagers, multifunctional proteins, bispecific or trispecific antibodies and chimeric antigen receptor (CAR) -related therapies.
In accordance with one embodiment of the present disclosure, therefore, provided is a single domain antibody or a polypeptide comprising the single domain antibody, wherein the single domain antibody has binding specificity to the B-cell maturation antigen (BCMA) protein and comprises the CDR1, CDR2 and CDR3 of any one of the antibodies A016, A022, A026, A034, A043, A052, A058, A060, A068, A075, A077, A081, A095, A098, A119, A130, A134, A135, A137, B010, B030, B077, B081, B109, B127, B133, B136, B168, B171, B172, B192, B231, B255, B270, B281, B282 and B208. The sequences of these antibodies are provided in Tables 1 and 3 and example CDR sequences (e.g., according to Kabat numbering) are shown in Tables 2 and 4.
In some embodiments, the CDR1, CDR2, and CDR3, respectively, comprise the amino acid sequences of: SEQ ID NO: 20, 21 and 22; SEQ ID NO: 23, 24 and 22; SEQ ID NO: 25, 26 and 27; SEQ ID NO: 28, 29 and 22; SEQ ID NO: 30, 31 and 32; SEQ ID NO: 33, 34 and 35;SEQ ID NO: 36, 37 and 38; SEQ ID NO: 39, 40 and 41; SEQ ID NO: 42, 43 and 44; SEQ ID NO: 45, 46 and 47; SEQ ID NO: 48, 49 and 50; SEQ ID NO: 51, 52 and 53; SEQ ID NO: 54, 55 and 56; SEQ ID NO: 57, 58 and 59; SEQ ID NO: 60, 61 and 62; SEQ ID NO: 63, 64 and 65; SEQ ID NO: 66, 67 and 68; SEQ ID NO: 69, 70 and 71; or SEQ ID NO: 72, 73 and 74.
In some embodiments, the CDR1, CDR2, and CDR3, respectively, comprise the amino acid sequences of: SEQ ID NO: 92, 93 and 94; SEQ ID NO: 95, 96 and 97; SEQ ID NO: 92, 98 and 99; SEQ ID NO: 100, 101 and 102; SEQ ID NO: 103, 104 and 94; SEQ ID NO: 105, 106 and 107; SEQ ID NO: 105, 108 and 109; SEQ ID NO: 92, 110 and 99; SEQ ID NO: 92, 111 and 99; SEQ ID NO: 103, 111 and 112; SEQ ID NO: 95, 96 and 113; SEQ ID NO: 103, 114 and 99; SEQ ID NO: 92, 115 and 116; SEQ ID NO: 103, 117 and 116; SEQ ID NO: 92, 118 and 116; SEQ ID NO: 92, 119 and 112; SEQ ID NO: 103, 124 and 112; or SEQ ID NO: 120, 121 and 122.
In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-19, or amino acid sequence having at least 80%sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1-19 while retaining the CDR of the corresponding sequence. In some embodiments, antibody or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-19.
Chimeric antigen receptors (CAR) , bispecific antibodies, trispecific antibodies, multifunctional proteins, and cells engineered to express any of these proteins are also provided, in various embodiments.
Methods of treatments and uses are also provided. In one embodiment, method of treating cancer in a patient in need thereof, comprising administering to the patient the antibody or polypeptide, the CAR, a polynucleotide encoding them, a cell engineered to express each, or an antibody-drug conjugate of the present disclosure. In some embodiments, the cancer is a hematological cancer, such as a BCMA-expressing B cell cancer (e.g., multiple myeloma) .
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the binding of the candidate antibodies from single-domain library to BCMA-HEK293, HEK293 and CHO-S cells as measured by FACS.
FIG. 2 shows the result of the non-specific binding test of the candidate antibodies from single-domain library to both HEK293 cells as measured by FACS.
FIG. 3 shows the result of the non-specific binding test of the candidate antibodies from single-domain library to both CHO-S cells as measured by FACS.
FIG. 4 shows the binding of the candidate antibodies from single-domain library to MM. 1S cells as measured by FACS.
FIG. 5 shows the FACS binding of the candidate antibodies from single-domain library to NCI-H929 cells.
FIG. 6 shows the FACS binding of the candidate antibodies from single-domain library to U266 cells.
FIG. 7 shows the FACS binding of the candidate antibodies from single-domain library to OPM-2 cells.
FIG. 8 shows the FACS binding of candidate antibodies from alpaca immune library to NCI-H929 cells.
FIG. 9 shows FACS binding of candidate antibodies from alpaca immune library to U266 cells.
FIG. 10 shows FACS binding of candidate antibodies from alpaca immune library to CHO-S cells.
FIG. 11 shows FACS binding of candidate antibodies from alpaca immune library to HEK293 cells.
FIG. 12 shows FACS binding of candidate antibodies from alpaca immune library to Jurkat cells.
DETAILED DESCRIPTION
Definitions
It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody, ” is understood to represent one or more antibodies. As such, the terms “a” (or “an” ) , “one or more, ” and “at least one” can be used interchangeably herein.
A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 98 %or 99 %) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Biologically equivalent polynucleotides are those having the above-noted specified percent homology and encoding a polypeptide having the same or similar biological activity.
The term “an equivalent nucleic acid or polynucleotide” refers to a nucleic acid having a nucleotide sequence having a certain degree of homology, or sequence identity, with the nucleotide sequence of the nucleic acid or complement thereof. A homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with or with the complement thereof. In one aspect, homologs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof. Likewise, “an equivalent polypeptide” refers to a polypeptide having a certain degree of homology, or sequence identity, with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide has one, two, three, four or five addition, deletion, substitution and their combinations thereof as compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (e.g., epitope-binding) or structure (e.g., salt-bridge) of the reference sequence.
As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.
The terms “antibody fragment” or “antigen-binding fragment” , as used herein, is a portion of an antibody such as F (ab’ ) 2, F (ab) 2, Fab’ , Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.
The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g., γl-γ4) . It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab’ and F (ab’ ) 2, Fd, Fvs, single-chain Fvs (scFv) , single-chain antibodies, disulfide-linked Fvs (sdFv) , fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies disclosed herein) . Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
A single-domain antibody (sdAb) , also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Nanobodies produced from camelids, alpacas and certain other animals are also referred to as VHH fragments. Like a whole antibody, a nanobody is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, single domain antibodies are much smaller than common antibodies (150–160 kDa) .
By “specifically binds” or “has specificity to, ” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B, ” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D. ”
As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
By “subject” or “individual” or “animal” or “patient” or “mammal, ” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
As used herein, phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.
In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” ( “CDR” ) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. MoI. Biol. 196: 901-917 (1987) , which are incorporated herein by reference in their entireties. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.
Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983) .
Anti-BCMA Single Domain Antibodies
From screening of a humanized single domain library, the instant inventors were able to identify a good number of single domain antibodies against the human BCMA protein, including A016, A022, A026, A034, A043, A052, A058, A060, A068, A075, A077, A081, A095, A098, A119, A130, A134, A135 and A137. These antibodies were subjected to cell-based binding tests, including with HEK293 cells engineered to express the human BCMA protein, BCMA-positive multiple myeloma human B lymphoblasts, HEK293, CHO-S as well as Jurkat cells Belantamab (GSK2857914) , a humanized IgG1 anti-BCMA monoclonal antibody, was used as benchmark antibody.
Similar screening and testing were also conducted with an immunized nanobody library, from which excellent binders were also selected. The top antibodies included B010, B030, B077, B081, B109, B127, B133, B136, B168, B171, B172, B192, B231, B255, B270, B281, B282 and B208, which are also considered suitable for further clinical development.
In accordance with one embodiment of the present disclosure, therefore, provided are single domain antibodies having binding specificity to a human B-cell maturation antigen (BCMA) protein. In one embodiment of the present disclosure, also provided are single domain antibodies and polypeptides that include such a single domain antibody.
In some embodiments, the single domain antibody includes a CDR1, a CDR2 and a CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of any of the identified antibodies A016, A022, A026, A034, A043, A052, A058, A060, A068, A075, A077, A081, A095, A098, A119, A130, A134, A135 and A137.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A016 (SEQ ID NO: 1) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 20, the CDR2 includes the amino acid sequence of SEQ ID NO: 21, and the CDR3 includes the amino acid sequence of SEQ ID NO: 22.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 1. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A016. Also provided, in some embodiments, are antibodies that compete with A016 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A022 (SEQ ID NO: 2) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 23, the CDR2 includes the amino acid sequence of SEQ ID NO: 24, and the CDR3 includes the amino acid sequence of SEQ ID NO: 22.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 2. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A022. Also provided, in some embodiments, are antibodies that compete with A022 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A026 (SEQ ID NO: 3) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 25, the CDR2 includes the amino acid sequence of SEQ ID NO: 26, and the CDR3 includes the amino acid sequence of SEQ ID NO: 27.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 3. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A026. Also provided, in some embodiments, are antibodies that compete with A034 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A034 (SEQ ID NO: 4) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 28, the CDR2 includes the amino acid sequence of SEQ ID NO: 29, and the CDR3 includes the amino acid sequence of SEQ ID NO: 22.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 4. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A034. Also provided, in some embodiments, are antibodies that compete with A034 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A043 (SEQ ID NO: 5) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 30, the CDR2 includes the amino acid sequence of SEQ ID NO: 31, and the CDR3 includes the amino acid sequence of SEQ ID NO: 32.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 5. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A043. Also provided, in some embodiments, are antibodies that compete with A043 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A052 (SEQ ID NO: 6) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 33, the CDR2 includes the amino acid sequence of SEQ ID NO: 34, and the CDR3 includes the amino acid sequence of SEQ ID NO: 35.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 6. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A052. Also provided, in some embodiments, are antibodies that compete with A052 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A058 (SEQ ID NO: 7) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 36, the CDR2 includes the amino acid sequence of SEQ ID NO: 37, and the CDR3 includes the amino acid sequence of SEQ ID NO: 38.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 7. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A058. Also provided, in some embodiments, are antibodies that compete with A058 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A060 (SEQ ID NO: 8) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 39, the CDR2 includes the amino acid sequence of SEQ ID NO: 40, and the CDR3 includes the amino acid sequence of SEQ ID NO: 41.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody includes the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 8. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A060. Also provided, in some embodiments, are antibodies that compete with A060 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A068 (SEQ ID NO: 9) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 42, the CDR2 includes the amino acid sequence of SEQ ID NO: 43, and the CDR3 includes the amino acid sequence of SEQ ID NO: 44.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 9. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A068. Also provided, in some embodiments, are antibodies that compete with A068 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A075 (SEQ ID NO: 10) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 45, the CDR2 includes the amino acid sequence of SEQ ID NO: 46, and the CDR3 includes the amino acid sequence of SEQ ID NO: 47.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 10. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A075. Also provided, in some embodiments, are antibodies that compete with A075 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A077 (SEQ ID NO: 11) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 48, the CDR2 includes the amino acid sequence of SEQ ID NO: 49, and the CDR3 includes the amino acid sequence of SEQ ID NO: 50.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 11. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A077. Also provided, in some embodiments, are antibodies that compete with A077 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A081 (SEQ ID NO: 12) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 51, the CDR2 includes the amino acid sequence of SEQ ID NO: 52, and the CDR3 includes the amino acid sequence of SEQ ID NO: 53.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 12. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A081. Also provided, in some embodiments, are antibodies that compete with A081 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A095 (SEQ ID NO: 13) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 54, the CDR2 includes the amino acid sequence of SEQ ID NO: 55, and the CDR3 includes the amino acid sequence of SEQ ID NO: 56.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 13. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A095. Also provided, in some embodiments, are antibodies that compete with A095 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A024 (SEQ ID NO: 14) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 57, the CDR2 includes the amino acid sequence of SEQ ID NO: 58, and the CDR3 includes the amino acid sequence of SEQ ID NO: 59.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody includes the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 14. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A024. Also provided, in some embodiments, are antibodies that compete with A024 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A119 (SEQ ID NO: 15) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 60, the CDR2 includes the amino acid sequence of SEQ ID NO: 61, and the CDR3 includes the amino acid sequence of SEQ ID NO: 62.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 15. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 15. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A119. Also provided, in some embodiments, are antibodies that compete with A119 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A130 (SEQ ID NO: 16) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 63, the CDR2 includes the amino acid sequence of SEQ ID NO: 64, and the CDR3 includes the amino acid sequence of SEQ ID NO: 65.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 16. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A130. Also provided, in some embodiments, are antibodies that compete with A130 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A134 (SEQ ID NO: 17) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 66, the CDR2 includes the amino acid sequence of SEQ ID NO: 67, and the CDR3 includes the amino acid sequence of SEQ ID NO: 68.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 17. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A134. Also provided, in some embodiments, are antibodies that compete with A134 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A135 (SEQ ID NO: 18) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 69, the CDR2 includes the amino acid sequence of SEQ ID NO: 70, and the CDR3 includes the amino acid sequence of SEQ ID NO: 71.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 18. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A135. Also provided, in some embodiments, are antibodies that compete with A135 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody A137 (SEQ ID NO: 19) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 72, the CDR2 includes the amino acid sequence of SEQ ID NO: 73, and the CDR3 includes the amino acid sequence of SEQ ID NO: 74.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 19. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as A137. Also provided, in some embodiments, are antibodies that compete with A137 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B010 (SEQ ID NO: 75) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 92, the CDR2 includes the amino acid sequence of SEQ ID NO: 93, and the CDR3 includes the amino acid sequence of SEQ ID NO: 94.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 75. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 75. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B010. Also provided, in some embodiments, are antibodies that compete with B010 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B030 (SEQ ID NO: 76) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 95, the CDR2 includes the amino acid sequence of SEQ ID NO: 96, and the CDR3 includes the amino acid sequence of SEQ ID NO: 97.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 76. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 76. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B030. Also provided, in some embodiments, are antibodies that compete with B030 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B077 (SEQ ID NO: 77) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 92, the CDR2 includes the amino acid sequence of SEQ ID NO: 98, and the CDR3 includes the amino acid sequence of SEQ ID NO: 99.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 77. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B077. Also provided, in some embodiments, are antibodies that compete with B077 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B081 (SEQ ID NO: 78) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 100, the CDR2 includes the amino acid sequence of SEQ ID NO: 101, and the CDR3 includes the amino acid sequence of SEQ ID NO: 102.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 78. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 78. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B081. Also provided, in some embodiments, are antibodies that compete with B081 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B109 (SEQ ID NO: 79) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 103, the CDR2 includes the amino acid sequence of SEQ ID NO: 104, and the CDR3 includes the amino acid sequence of SEQ ID NO: 94.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 79. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 79. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B109. Also provided, in some embodiments, are antibodies that compete with B109 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B127 (SEQ ID NO: 80) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 105, the CDR2 includes the amino acid sequence of SEQ ID NO: 106, and the CDR3 includes the amino acid sequence of SEQ ID NO: 107.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 80. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 80. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B127. Also provided, in some embodiments, are antibodies that compete with B127 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B133 (SEQ ID NO: 81) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 105, the CDR2 includes the amino acid sequence of SEQ ID NO: 108, and the CDR3 includes the amino acid sequence of SEQ ID NO: 109.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 81. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 81. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B133. Also provided, in some embodiments, are antibodies that compete with B133 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B136 (SEQ ID NO: 82) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 92, the CDR2 includes the amino acid sequence of SEQ ID NO: 110, and the CDR3 includes the amino acid sequence of SEQ ID NO: 99.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 82. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 82. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B136. Also provided, in some embodiments, are antibodies that compete with B136 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B168 (SEQ ID NO: 83) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 92, the CDR2 includes the amino acid sequence of SEQ ID NO: 111, and the CDR3 includes the amino acid sequence of SEQ ID NO: 99.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 83. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 83. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B168. Also provided, in some embodiments, are antibodies that compete with B168 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B171 (SEQ ID NO: 84) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 103, the CDR2 includes the amino acid sequence of SEQ ID NO: 111, and the CDR3 includes the amino acid sequence of SEQ ID NO: 112.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 84. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 84. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B171. Also provided, in some embodiments, are antibodies that compete with B171 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B172 (SEQ ID NO: 85) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 95, the CDR2 includes the amino acid sequence of SEQ ID NO: 96, and the CDR3 includes the amino acid sequence of SEQ ID NO: 113.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 85. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 85. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B172. Also provided, in some embodiments, are antibodies that compete with B172 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B192 (SEQ ID NO: 86) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 103, the CDR2 includes the amino acid sequence of SEQ ID NO: 114, and the CDR3 includes the amino acid sequence of SEQ ID NO: 99.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 86. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 86. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B192. Also provided, in some embodiments, are antibodies that compete with B192 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B231 (SEQ ID NO: 87) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 92, the CDR2 includes the amino acid sequence of SEQ ID NO: 115, and the CDR3 includes the amino acid sequence of SEQ ID NO: 116.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 87. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 87. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B231. Also provided, in some embodiments, are antibodies that compete with B231 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B255 (SEQ ID NO: 88) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 103, the CDR2 includes the amino acid sequence of SEQ ID NO: 117, and the CDR3 includes the amino acid sequence of SEQ ID NO: 116.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 88. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 88. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B255. Also provided, in some embodiments, are antibodies that compete with B255 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B270 (SEQ ID NO: 89) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 92, the CDR2 includes the amino acid sequence of SEQ ID NO: 118, and the CDR3 includes the amino acid sequence of SEQ ID NO: 116.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 89. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 89. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B270. Also provided, in some embodiments, are antibodies that compete with B270 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B281 (SEQ ID NO: 90) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 92, the CDR2 includes the amino acid sequence of SEQ ID NO: 119, and the CDR3 includes the amino acid sequence of SEQ ID NO: 112.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 90. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 90. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B281. Also provided, in some embodiments, are antibodies that compete with B281 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B282 (SEQ ID NO: 123) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 103, the CDR2 includes the amino acid sequence of SEQ ID NO: 124, and the CDR3 includes the amino acid sequence of SEQ ID NO: 112.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 123. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 123. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B282. Also provided, in some embodiments, are antibodies that compete with B282 in binding to BCMA.
In one embodiment, the single domain antibody includes the CDR1, CDR2 and CDR3 of antibody B208 (SEQ ID NO: 91) . In some embodiments, the CDR1 includes the amino acid sequence of SEQ ID NO: 120, the CDR2 includes the amino acid sequence of SEQ ID NO: 121, and the CDR3 includes the amino acid sequence of SEQ ID NO: 122.
In some embodiments, the antibody includes the amino acid sequence of SEQ ID NO: 91. In some embodiments, the antibody includes the recited CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99%sequence identity to SEQ ID NO: 91. Also provided, in some embodiments, are antibodies that bind to the same epitope on BCMA as B208. Also provided, in some embodiments, are antibodies that compete with B208 in binding to BCMA.
A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
It will also be understood by one of ordinary skill in the art that antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%identical to the starting sequence.
Antibodies, variants, or derivatives thereof of the disclosure include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to the epitope. For example, but not by way of limitation, the antibodies can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.
Antibody-Drug Conjugates
In some embodiments, the antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
In one embodiment, the antibodies of the disclosure are covalently attached to a drug moiety. The drug moiety may be, or be modified to include, a group reactive with a conjugation point on the antibody. For example, a drug moiety can be attached by alkylation (e.g., at the epsilon-amino group lysines or the N-terminus of antibodies) , reductive amination of oxidized carbohydrate, transesterification between hydroxyl and carboxyl groups, amidation at amino groups or carboxyl groups, and conjugation to thiols.
In some embodiments, the number of drug moieties, p, conjugated per antibody molecule ranges from an average of 1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from an average of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In other embodiments, p is an average of 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, p ranges from an average of about 1 to about 20, about 1 to about 10, about 2 to about 10, about 2 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2. In some embodiments, p ranges from about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4 or about 2 to about 3.
For example, when chemical activation of the protein results in formation of free thiol groups, the protein may be conjugated with a sulfhydryl reactive agent. In one aspect, the agent is one which is substantially specific for free thiol groups. Such agents include, for example, malemide, haloacetamides (e.g., iodo, bromo or chloro) , haloesters (e.g., iodo, bromo or chloro) , halomethyl ketones (e.g., iodo, bromo or chloro) , benzylic halides (e.g., iodide, bromide or chloride) , vinyl sulfone and pyridylthio.
The drug can be linked to the antibody or fragment by a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. A cleavable linker is typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) , a phenylalanine-lysine (phe-lys) linker, or maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) linker. Another linker is Sulfosuccinimidyl-4- [N-maleimidomethyl] cyclohexane-1-carboxylate (smcc) . Sulfo-smcc conjugation occurs via a maleimide group which reacts with sulfhydryls (thiols, -SH) , while its Sulfo-NHS ester is reactive toward primary amines (as found in Lysine and the protein or peptide N-terminus) . Yet another linker is maleimidocaproyl (mc) . Other suitable linkers include linkers hydrolyzable at a specific pH or a pH range, such as a hydrazone linker. Additional suitable cleavable linkers include disulfide linkers. The linker may be covalently bound to the antibody to such an extent that the antibody must be degraded intracellularly in order for the drug to be released e.g. the mc linker and the like.
A linker can include a group for linkage to the antibody. For example, linker can include an amino, hydroxyl, carboxyl or sulfhydryl reactive groups (e.g., malemide, haloacetamides (e.g., iodo, bromo or chloro) , haloesters (e.g., iodo, bromo or chloro) , halomethyl ketones (e.g., iodo, bromo or chloro) , benzylic halides (e.g., iodide, bromide or chloride) , vinyl sulfone and pyridylthio) .
In some embodiments, the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressive agent, a radioisotope, a toxin, or the like. The conjugate can be used for inhibiting the multiplication of a tumor cell or cancer cell, causing apoptosis in a tumor or cancer cell, or for treating cancer in a patient. The conjugate can be used accordingly in a variety of settings for the treatment of animal cancers. The conjugate can be used to deliver a drug to a tumor cell or cancer cell. Without being bound by theory, in some embodiments, the conjugate binds to or associates with a cancer cell expressing BCMA, and the conjugate and/or drug can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis.
Once inside the cell, one or more specific peptide sequences within the conjugate (e.g., in a linker) are hydrolytically cleaved by one or more tumor-cell or cancer-cell-associated proteases, resulting in release of the drug. The released drug is then free to migrate within the cell and induce cytotoxic or cytostatic or other activities. In some embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, and the drug subsequently penetrates the cell, or acts at the cell surface.
Examples of drug moieties or payloads are selected from the group consisting of DM1 (maytansine, N2’ -deacetyl-N2’ - (3-mercapto-1-oxopropyl) -or N2’ -deacetyl-N2’ - (3-mercapto-1-oxopropyl) -maytansine) , mc-MMAD (6-maleimidocaproyl-monomethylauristatin-D or N-methyl-L-valyl-N- [ (1S, 2R) -2-methoxy-4- [ (2S) -2- [ (1R, 2R) -1-methoxy-2-methyl-3-oxo-3- [ [ (1S) -2-phenyl-1- (2-thiazolyl) ethyl] amino] propyl] -1-pyr rolidinyl] -1- [ (1S) -1-methylpropyl] -4-oxobutyl] -N-methyl- (9Cl) -L-valinamide) , mc-MMAF (maleimidocaproyl-monomethylauristatin F or N- [6- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) -1-oxohexyl] -N-methyl-L-valyl-L-valyl- (3R, 4S, 5S) -3-methoxy-5-methyl-4- (methylamino) heptanoyl- (αR, βR,2S) -β-methoxy-α-methyl-2-pyrrolidinepropanoyl-L-phenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-ValcCit- (p-aminobenzyloxycarbonyl) -monomethylauristatin E or N- [ [ [4- [ [N- [6- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) -1-oxohexyl] -L-valyl-N5- (aminocarbonyl) -L-ornithyl] amino] phenyl] methoxy] carbonyl] -N-meth yl-L-valyl-N- [ (1S, 2R) -4- [ (2S) -2- [ (1R, 2R) -3- [ [ (1R, 2S) -2-hydroxy-1-methyl-2-phenylethyl] amino] -1-methoxy-2-methyl-3-oxopropyl] -1-pyrrolidinyl] -2-methoxy-1- [ (1S) -1-methylpropyl] -4-oxobutyl] -N-methyl-L-valinamide) . DM1 is a derivative of the tubulin inhibitor maytansine while MMAD, MMAE, and MMAF are auristatin derivatives. In some embodiments, the drug moiety is selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE. In some embodiments, the drug moiety is a maytansinoid or an auristatin.
The antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.
The antibodies can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
The antibodies can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) . Techniques for conjugating various moieties to an antibody are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy” , in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds. ) , pp. 243-56 (Alan R. Liss, Inc. (1985) ; Hellstrom et al., “Antibodies For Drug Delivery” , in Controlled Drug Delivery (2nd Ed. ) , Robinson et al., (eds. ) , Marcel Dekker, Inc., pp. 623-53 (1987) ; Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review” , in Monoclonal Antibodies ‘84: Biological And Clinical Applications, Pinchera et al. (eds. ) , pp. 475-506 (1985) ; “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy” , in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds. ) , Academic Press pp. 303-16 (1985) , and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates” , Immunol. Rev. (52: 119-58 (1982) ) .
Multi-functional Molecules
Multi-functional molecules are provided that include an antibody specific to BCMA, such as those disclosed herein, and one or more antibodies or antigen-binding fragments having specificity to a second antigen or a second epitope on BCMA.
In some embodiments, the second antigen is a protein expressed on an immune cell, such as a T cell, a B cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a phagocyte, a natural killer cell, an eosinophil, a basophil, and a mast cell.
In some embodiments, the second antigen is GPRC5D, CD123, CS1, CD20, CD19, CD22, CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.
In some embodiments, the second specificity is to a different epitope on BCMA, or amino acid residues on BCMA that are different from or overlapping with those bound by the antibodies of the instant disclosure.
Different formats of bispecific antibodies are also provided. In some embodiments, the second fragment can be selected from a Fab fragment, a single-chain variable fragment (scFv) , or a single-domain antibody. In some embodiments, the bispecific antibody further includes a Fc fragment.
Bifunctional molecules that include not just antibody or antigen binding fragment are also provided. As a tumor antigen targeting molecule, an antibody or antigen-binding fragment specific to BCMA, such as those described here, can be combined with an immune cytokine or ligand optionally through a peptide linker. The linked immune cytokines or ligands include, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL. Such bi-functional molecules can combine the immune checkpoint blocking effect with tumor site local immune modulation.
Also provided are a particular bispecific or bifunctional antibody known as T-cell engager. T cell engagers having a specificity to the T cell (e.g., CD3 bispecific antibodies) provide a targeted immuno-oncology platform that connects patients’ own T cells to malignant cells. Such bispecific T cell engagers ensure direct connection of the T-cell to the cancer cell and thus can enable T-cell activation, which results in cytotoxic activity directed to the cancer cell.
In one embodiments, therefore, the present disclosure provides a T-cell engager that includes an antibody or polypeptide of the present disclosure. T-cell engagers can come with different molecular designs and can be particularly useful for the treatment of liquid or solid tumors. In some embodiments, the T cell engager is bispecific, which can target BCMA and GPRC5D. In some embodiments, the T cell engager is trispecific, which can target BCMA and GPRC5D, and yet another epitope on BCMA or another tumor antigen.
Chimeric Antigen Receptors
Also provided, in one embodiment, is a chimeric antigen receptor (CAR) that includes the antibody of the present disclosure as a targeting unit. In some embodiments, the CAR includes one or more antibodies or fragments thereof of the present disclosure, a transmembrane domain, a costimulatory domain, and a CD3ξ intracellular domain.
A transmembrane domain can be designed to be fused to the extracellular domain which includes the antibody or fragment, optionally through a hinge domain. It can similarly be fused to an intracellular domain, such as a costimulatory domain. In some embodiments, the transmembrane domain can include the natural transmembrane region of a costimulatory domain (e.g., the TM region of a CD28T or 4-1BB employed as a costimulatory domain) or the natural transmembrane domain of a hinge region (e.g., the TM region of a CD8 alpha or CD28T employed as a hinge domain) .
In some embodiments, the transmembrane domain can include a sequence that spans a cell membrane, but extends into the cytoplasm of a cell and/or into the extracellular space. For example, a transmembrane can include a membrane-spanning sequence which itself can further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm of a cell, and/or the extracellular space. Thus, a transmembrane domain includes a membrane-spanning region, yet can further comprise an amino acid (s) that extend beyond the internal or external surface of the membrane itself; such sequences can still be considered to be a “transmembrane domain” .
In some embodiments, the transmembrane domain is fused to the cytoplasmic domain through a short linker. Optionally, the short peptide or polypeptide linker, preferably between 2 and 10 amino acids in length can form the linkage between the transmembrane domain and a proximal cytoplasmic signaling domain of the chimeric receptor. A glycine-serine doublet (GS) , glycine-serine-glycine triplet (GSG) , or alanine-alanine-alanine triplet (AAA) provides a suitable linker.
In some embodiments, the CAR further includes a costimulatory domain. In some embodiments, the costimulatory domain is positioned between the transmembrane domain and an activating domain. Example costimulatory domains include, but are not limited to, CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8, CD11a (ITGAL) , CD11b (ITGAM) , CD11c (ITGAX) , CD11d (ITGAD) , CD18 (ITGB2) , CD19 (B4) , CD27 (T FRSF7) , CD28, CD28T, CD29 (ITGB1) , CD30 (TNFRSF8) , CD40 (TNFRSF5) , CD48 (SLAMF2) , CD49a (ITGA1) , CD49d (ITGA4) , CD49f (ITGA6) , CD66a (CEACAM1) , CD66b (CEACAM8) , CD66c (CEACAM6) , CD66d (CEACAM3) , CD66e (CEACAM5) , CD69 (CLEC2) , CD79A (B-cell antigen receptor complex-associated alpha chain) , CD79B (B-cell antigen receptor complex-associated beta chain) , CD84 (SLAMF5) , CD96 (Tactile) , CD 100 (SEMA4D) , CD 103 (ITGAE) , CD134 (OX40) , CD137 (4-1BB) , CD150 (SLAMF1) , CD158A (KIR2DL1) , CD158B1 (KIR2DL2) , CD158B2 (KIR2DL3) , CD158C (KIR3DP1) , CD158D (KIRDL4) , CD158F1 (KIR2DL5A) , CD158F2 (KIR2DL5B) , CD158K (KTR3DL2) , CD160 (BY55) , CD162 (SELPLG) , CD226 (DNAM1) , CD229 (SLAMF3) , CD244 (SLAMF4) , CD247 (CD3-zeta) , CD258 (LIGHT) , CD268 (BAFFR) , CD270 (T FSF14) , CD272 (BTLA) , CD276 (GPRC5D) , CD279 (PD-1) , CD314 (KG2D) , CD319 (SLAMF7) , CD335 (K-p46) , CD336 (K-p44) , CD337 (K-p30) , CD352 (SLAMF6) , CD353 (SLAMF8) , CD355 (CRTAM) , CD357 (TNFRSF 18) , inducible T cell co-stimulator (ICOS) , LFA-1 (CD 1 la/CD 18) , KG2C, DAP-10, ICAM-1, Kp80 (KLRF1) , IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL) , SLP-76 (LCP2) , PAG1/CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, a Toll ligand receptor, and fragments or combinations thereof.
In some embodiments, the cytoplasmic portion of the CAR also includes a signaling/activation domain. In one embodiment, the signaling/activation domain is the CD3ξ domain, or is an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98%or 99%sequence identity to the CD3ξ domain.
Bispecific and bicistronic CARs are also provided. For a bispecific CAR, the CAR protein further includes a second antigen-binding fragment (e.g., scFv) that binds a second target. Non-limiting examples of the second target include GPRC5D, CD123, CS1, CD20, CD19, CD22, CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, and VEGF. A bicistronic CAR includes two CAR molecules that may be expressed by the same vector encoding both. Both CAR molecules can be expressed in a target cell, such as a T cell.
Cells that enclose the CAR or the encoding nucleotides are also provided in the present disclosure. A suitable cell can be used, that is transduced with a vector that encodes, or put in contact with, an CAR that includes an anti-BCMA antibody of the present disclosure (or alternatively engineered to express an anti-BCMA antibody of the present disclosure) . The cell (e.g., T cell, NK cell, monocyte, macrophage) can be, for instance, a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, a gamma delta T cell, or the combination thereof, without limitation.
Polynucleotides, mRNA, and Methods of Expressing or Preparing Antibodies
The present disclosure also provides polynucleotides or nucleic acid molecules encoding the antibodies, variants or derivatives thereof of the disclosure, or the CAR. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.
In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into a target cell for expressing the antibody or fragment thereof.
mRNAs may be synthesized according to any of a variety of known methods. For example, the mRNAs may be synthesized via in vitro transcription (IVT) . Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase) , DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application.
In some embodiments, for the preparation of antibody-coding mRNA, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired antibody encoding (e.g., heavy chain or light chain encoding) mRNA and a termination signal.
Desired antibody encoding (e.g., heavy chain or light chain encoding) mRNA sequence may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., a desired heavy chain or light chain sequence) , a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.
The mRNA may be synthesized as unmodified or modified mRNA. Typically, mRNAs are modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA. A modified mRNA can thus include, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, antibody encoding mRNAs (e.g., heavy chain and light chain encoding mRNAs) may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A) , guanine (G) ) or pyrimidines (thymine (T) , cytosine (C) , uracil (U) ) , and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2, 6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2, 2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil) , dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5- (carboxyhydroxymethyl) -uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5’ -methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v) , 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g. from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosure of which is included here in its full scope by reference.
In some embodiments, the mRNAs (e.g., heavy chain and light chain encoding mRNAs) may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5’ -O- (1-thiophosphate) ) , boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.
In some embodiments, the mRNAs (e.g., heavy chain and light chain encoding mRNAs) may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2’ -deoxy-2’ -fluoro-oligoribonucleotide (2’ -fluoro-2’ -deoxycytidine 5’ -triphosphate, 2’ -fluoro-2’ -deoxyuridine 5’ -triphosphate) , 2’ -deoxy-2’ -deamine-oligoribonucleotide (2’ -amino-2’ -deoxycytidine 5’ -triphosphate, 2’ -amino-2’ -deoxyuridine 5’ -triphosphate) , 2’ -O-alkyloligoribonucleotide, 2’ -deoxy-2’ -C-alkyloligoribonucleotide (2’ -O-methylcytidine 5’ -triphosphate, 2’ -methyluridine 5’ -triphosphate) , 2’ -C-alkyloligoribonucleotide, and isomers thereof (2’ -aracytidine 5’ -triphosphate, 2’ -arauridine 5’ -triphosphate) , or azidotriphosphates (2’ -azido-2’ -deoxycytidine 5’ -triphosphate, 2’ -azido-2’ -deoxyuridine 5’ -triphosphate) .
In some embodiments, the mRNAs (e.g., heavy chain and light chain encoding mRNAs) may contain modifications of the bases of the nucleotides (base modifications) . A modified nucleotide which contains a base modification is also called a base-modified nucleotide. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine riboside 5’ -triphosphate, 2-aminoadenosine 5’ -triphosphate, 2-thiocytidine 5’ -triphosphate, 2-thiouridine 5’ -triphosphate, 4-thiouridine 5’ -triphosphate, 5-aminoallylcytidine 5’ -triphosphate, 5-aminoallyluridine 5’ -triphosphate, 5-bromocytidine 5’ -triphosphate, 5-bromouridine 5’ -triphosphate, 5-iodocytidine 5’ -triphosphate, 5-iodouridine 5’ -triphosphate, 5-methylcytidine 5’ -triphosphate, 5-methyluridine 5’ -triphosphate, 6-azacytidine 5’ -triphosphate, 6-azauridine 5’ -triphosphate, 6-chloropurine riboside 5’ -triphosphate, 7-deazaadenosine 5’ -triphosphate, 7-deazaguanosine 5’ -triphosphate, 8-azaadenosine 5’ -triphosphate, 8-azidoadenosine 5’ -triphosphate, benzimidazole riboside 5’ -triphosphate, N1-methyladenosine 5’ -triphosphate, N1-methylguanosine 5’ -triphosphate, N6-methyladenosine 5’ -triphosphate, O6-methylguanosine 5’ -triphosphate, pseudouridine 5’ -triphosphate, puromycin 5’ -triphosphate or xanthosine 5’ -triphosphate.
Typically, mRNA synthesis includes the addition of a “cap” on the N-terminal (5’ ) end, and a “tail” on the C-terminal (3’ ) end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.
Thus, in some embodiments, the mRNAs (e.g., heavy chain and light chain encoding mRNAs) include a 5’ cap structure. A 5’ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5’ 5’ 5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G (5’ ) ppp (5’ (A, G (5’ ) ppp (5) A and G (5) ppp (5’ ) G.
In some embodiments, the mRNAs (e.g., heavy chain and light chain encoding mRNAs) include a 3’ poly (A) tail structure. A poly-A tail on the 3’ terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 125 adenosine nucleotides, 10 to 100 adenosine nucleotides, about 10 to 75 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides) . In some embodiments, antibody encoding mRNAs (e.g., heavy chain and light chain encoding mRNAs) include a 3’ poly (C) tail structure. A suitable poly-C tail on the 3’ terminus of mRNA typically includes about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides) . The poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.
In some embodiments, the mRNAs (e.g., heavy chain and light chain encoding mRNAs) include a 5’ and/or 3’ untranslated region. In some embodiments, a 5’ untranslated region includes one or more elements that affect an mRNA’s stability or translation, for example, an iron responsive element. In some embodiments, a 5’ untranslated region may be between about 50 and 500 nucleotides in length (e.g., about 50 and 400 nucleotides in length, about 50 and 300 nucleotides in length, about 50 and 200 nucleotides in length, or about 50 and 100 nucleotides in length) .
In some embodiments, a 5’ region of an mRNA (e.g., heavy chain and light chain encoding mRNAs) includes a sequence encoding a signal peptide, such as those described herein. In particular embodiments, a signal peptide derived from human growth hormone (hGH) is incorporated in the 5’ region. Typically, a signal peptide encoding sequence is linked, directly or indirectly, to the heavy chain or light chain encoding sequence at the N-terminus.
The present technology may be used to deliver any antibody known in the art and antibodies that can be produced against desired antigens using standard methods. The present invention may be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, or bi-specific antibodies.
Methods of making antibodies are well known in the art and described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. patents: 6,150,584; 6,458,592; 6,420,140 which are incorporated by reference in their entireties.
Treatment and Uses
As described herein, the antibodies, variants, antibody-drug conjugates, chimeric antigen receptors (CAR) and CAR cells, encoding polynucleotides or derivatives of the present disclosure may be used in certain treatment and diagnostic methods.
The present disclosure is further directed to antibody-based therapies which involve administering the antibodies or fragments, antibody-drug conjugates, chimeric antigen receptors (CAR) and CAR cells, encoding polynucleotides or derivatives of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein. Therapeutic molecules or cells of the disclosure include, but are not limited to, antibodies of the disclosure (including variants and derivatives thereof as described herein) , antibody-drug conjugates, chimeric antigen receptors (CAR) and CAR cells, and nucleic acids or polynucleotides encoding antibodies of the disclosure (including variants and derivatives thereof as described herein) .
The molecules or cells of the disclosure can also be used to treat or inhibit cancer. As provided above, BCMA can be overexpressed in tumor cells, in particular hematological cancers. In some embodiments, the cancer is a BCMA-expressing B cell cancer, such as multiple myeloma.
Accordingly, in some embodiments, provided are methods for treating a cancer in a patient in need thereof. The method, in one embodiment, entails administering to the patient an effective amount of the molecules or cells of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient over-express BCMA.
Cellular therapies, such as chimeric antigen receptor (CAR) T-cell therapies, are also provided in the present disclosure. A suitable cell can be used, that is transduced with a vector that encodes, or put in contact with, a CAR that includes an anti-BCMA antibody of the present disclosure (or alternatively engineered to express an anti-BCMA antibody of the present disclosure) . Upon such contact or engineering, the cell can then be introduced to a cancer patient in need of treatment. The cancer patient may have a cancer of any of the types as disclosed herein. The cell (e.g., T cell, NK cell, monocyte, macrophage) can be, for instance, a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, a gamma delta T, or the combination thereof, without limitation.
In some embodiments, the cell was isolated from the cancer patient him-or her-self. In some embodiments, the cell was provided by a donor or from a cell bank. When the cell is isolated from the cancer patient, undesired immune reactions can be minimized.
Non-limiting examples of cancers include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of gastric, pancreatic, esophageal, ovarian, and lung cancers.
Additional diseases or conditions associated with increased cell survival, that may be treated, prevented, diagnosed and/or prognosed with the antibodies or variants, or derivatives thereof of the disclosure include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) ) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia) ) , polycythemia vera, lymphomas (e.g., Hodgkin’s disease and non-Hodgkin’s disease) , multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyo sarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm’s tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma. A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antibodies, variant or derivative thereof used, the patient’s age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.
Multiple myeloma (MM) is the second most common hematological malignancy and constitutes 2%of all cancer deaths. MM is a heterogeneous disease and caused by mostly by chromosome translocations inter alia t (l 1 ; 14) , t (4; 14) , t (8; 14) , del (13) , del (17) (Drach et al., (1998) Blood 92 (3) : 802-809; Gertz et al., (2005) Blood 106 (8) : 2837-2840; Facon et al., (2001) Blood 97 (6) : 1566-1571) . MM-affected patients may experience a variety of disease-related symptoms due to, bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and the psychosocial burden of a cancer diagnosis. As of 2006, the 5-year relative survival rate for MM was approximately 34%highlighting that MM is a difficult-to-treat disease where there are currently no curative options.
Methods of administration of the antibody or fragment include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptides or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc. ) and may be administered together with other biologically active agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch) , buccally, or as an oral or nasal spray.
The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.
Administration can be systemic or local. In addition, it may be desirable to introduce the antibodies of the disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
It may be desirable to administer the antigen-binding polypeptides or compositions of the disclosure locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction, with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the disclosure, care must be taken to use materials to which the protein does not absorb.
The amount of the antibodies of the disclosure which will be effective in the treatment, inhibition and prevention of an inflammatory, immune or malignant disease, disorder or condition can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, disorder or condition, and should be decided according to the judgment of the practitioner and each patient’s circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
As a general proposition, the dosage administered to a patient of the antibodies of the present disclosure is typically 0.001 mg/kg to 100 mg/kg of the patient’s body weight, between 0.01 mg/kg and 20 mg/kg of the patient’s body weight, or 0.5 mg/kg to 10 mg/kg of the patient’s body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the disclosure may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
In an additional embodiment, the compositions of the disclosure are administered in combination with cytokines. Cytokines that may be administered with the compositions of the disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.
In additional embodiments, the compositions of the disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
Compositions
The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody, antibody-drug conjugates, chimeric antigen receptors (CAR) and CAR cells, encoding polynucleotides, or derivatives and an acceptable carrier. In some embodiments, the composition further includes a second anticancer agent (e.g., an immune checkpoint inhibitor) .
In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Further, a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences by E. W. Martin, incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
In an embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The compounds of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
EXAMPLES
Example 1: Screening of Anti-BCMA Single Domain Antibodies
This example demonstrates the screening for positive clones through panning of a synthetic, humanized single domain library and primary screening, where Hu-BCMA-ECD-His, Hu-BCMA-ECD-Fc, Cyno-BCMA-ECD-His, Cyno-BCMA-ECD-Fc antigens and Hu-BCMA-HEK293, NCI-H929 were used as antigens (or controls) .
Materials and Methods
Panning of Humanized Single Domain Library (Liquid-phase Method) and coated with 
Kingfisher
Depletion of non-specific phages: after being diluted and blocked with 2.5%BSA solution, the phage suspensions of the prepared humanized single domain library were incubated with Dynabeads, and depleted phages without non-specific binding were collected after incubation.
Dynabeads blocking: fresh prepared magnetic beads were blocked with 2.5%BSA.
Positive panning: coated beads were incubated with depleted phages, and further washed according to the Liquid-phase magnetic bead screening system method.
Elution: beads were eluted by trypsin and the eluate containing the phages was collected.
Infection and cultivation: collected phages were mixed with the logarithmic phase SS320 cells and incubated for 30 min at 37℃. After the incubation, spreading of infected cells on the 2YT-Car+-Tet+ plate was performed and incubation was conducted at 37℃ overnight in an incubator.
Titer tests: the eluted phage solution was diluted in 10-fold gradient manner and incubated with logarithmic phase SS320 cells, after which 2 μL of mixed solution was spread on the plate and incubated at 37℃ overnight in the incubator.
Statistics: three rounds of panning were performed with 3-fold gradient decreasing of antigen concentration, and antibody clones with high affinity were obtained. The enrichment of positive clones was evaluated based on the calculation of input and output titers.
Panning of Humanized Single Domain Library (Immunotube Method)
Blocking: prepared humanized single-domain library phage suspensions were blocked with 5%PBSM.
Immunotube coating and blocking: the diluted antigens were added into the immunotubes and coat overnight at 4℃, then washed and blocked with 5%PBSM.
Positive panning: phage suspensions were incubated in antigen-coated immunotubes. Binding and subsequent wash processes were conducted according to the immunotube screening system method.
Elution: immunotubes were eluted by trypsin and the eluate containing the phages was collected.
Infection and cultivation: collected phages were mixed with the logarithmic phase SS320 cells and incubated for 30 min at 37℃. After the incubation, spreading of infected cells on the 2YT-Car+-Tet+ plate was performed and incubation was conducted at 37℃ overnight in an incubator.
Titer tests: the eluted phage solution was diluted in 10-fold gradient manner and incubated with logarithmic phase SS320 cells, after which 2 μL of mixed solution was spread on the plate and incubated at 37℃ overnight in the incubator.
Statistics: three rounds of panning were performed with 3-fold gradient decreasing of antigen concentration, and antibody clones with high affinity were obtained. The enrichment of positive clones was evaluated based on the calculation of input and output titers.
Panning of Humanized Single Domain Library (Cell-based Panning)
Phage blocking: the prepared phage libraries were blocked with 5%PBSM at room temperature on the four-dimensional rotary instrument for 1 h.
Incubation
1) Negative panning: blocked phage suspensions were added into the negative cell solution, and then the mixture was placed on orbital shaker at (room temperature, 80 rpm, 1 hour) . Notice: phage suspension was added carefully in order not to wash out the cell.
2) Positive panning: negatively screened phages were added into the corresponding positive cell bottles using a disposable sucker and the mixture was incubated on orbital shaker (room temperature, 80 rpm, 2 hours) .
Washing: supernatant was discarded and 5%FBS-PBS solution was added into the bottles. To make sure that all cells would be covered by liquid, a short mix process was performed on the orbital shaker with a rotating speed of 80 rpm for 1 minute. The washing procedure was repeated for serval times, which was determined based on the round of panning.
Elution: 1 mL 100 mM glycine-HCl solution (pH 2.2) were added to bottles, and the mixed samples were further incubated at room temperature on a shaking at 80 rpm for 10 min. Additional 70 μL Tris-base solutions (1 M) were added to neutralize and the pH was adjusted to 7.0-7.2 range. The eluate containing the positive phages was collected a 15 mL centrifuge tube for later usage.
Infection and cultivation: collected phages were mixed with the logarithmic phase SS320 cells and incubated for 30 min at 37℃. After the incubation, spreading of infected cells on the 2YT-Car+-Tet+ plate was performed and incubation was conducted at 37℃ overnight in an incubator.
Titer tests: the eluted phage solution was diluted in 10-fold gradient manner and incubated with logarithmic phase SS320 cells, after which 2 μL of mixed solution was spread on the plate and incubated at 37℃ overnight in the incubator.
Statistics: multiple rounds of panning were performed with 3-fold gradient decreasing of antigen concentration, and antibody clones with high affinity were obtained. The enrichment of positive clones was evaluated based on the calculation of input and output titers.
Positive Antibody Signal Validation
The expression supernatant of phage pools after 3 rounds of screening was diluted in 5-fold gradient with 5%PBSM, and ELISA was performed.
ELISA method
1) Coating: 2 μg/mL antigen Hu-BCMA-ECD-His, Cyno-BCMA-ECD-His, 30 μL/well, overnight at 4℃, wash the plates 3 times with PBST.
2) Blocking: Block with 5%PBSM at room temperature for 1 h, wash the plates 3 times with PBST.
3) Primary antibody: Add the above VHH or Phage expression supernatant after gradient dilution, 30 μL/well, place at room temperature for 1 h, then wash the plate 3 times with PBST.
4) Secondary antibody: Add 1: 5000 diluted secondary antibody Anti-Flag-HRP to VHH supernatant, dilute with PBS, 30 μL/well, place at room temperature for 1 h, wash the plate 6 times with PBST; Add 1: 20000 diluted secondary antibody Anti-M13-HRP to Phage, dilute with 5%PBSM, 30 μL/well, place at room temperature for 1 h, then wash the plate 6 times with PBST.
5) Stopping: Add TMB, 30 μL/well, develop the color for 5 min to 10 min at room temperature, add 2 M stop solution to stop the reaction, 30 μL/well. The OD values of each well were read at 450 nm with a microplate reader.
Quantitative ELISA method
1) Coating: Dilute Anti-Flag to 1 μg/mL with PBS, 30 μL/well, place at room temperature for 16 h, wash the plate 3 times with PBST.
2) Blocking: Block with 5%PBSM at room temperature for 1 h, wash the plates 3 times with PBST.
3) Primary antibody: Add gradient diluted VHH supernatant 30 μL/well, place at room temperature for 1 h, then wash the plate 3 times with PBST.
4) Secondary antibody: Add 30 μL of diluted secondary antibody Anti-VHH-HRP (1: 7000) , place at room temperature for 1 h, then wash the plate 6 times with PBST.
5) Stopping: Add TMB, 30 μL/well, develop the color for 5 -20 min at room temperature, add 30 μL/well of 2 M stop solution to stop the reaction. The OD values of each well were read at 450 nm with a microplate reader.
Monoclonal primary screening
Monoclonal Phage supernatant was separated by centrifugation for primary screening ELISA.
ELISA method
1) Coating: 2 μg/mL Hu-BCMA-ECD-His, Cyno-BCMA-ECD-His, 30 μL/well, overnight at 4℃, wash the plates 3 times with PBST.
2) Blocking: Block with 5%PBSM at room temperature for 1 h, then wash the plates 3 times with PBST.
3) Primary antibody: Add gradient diluted Phage supernatant 30 μL/well, place at room temperature for 1 h, wash the plate 3 times with PBST.
4) Secondary antibody: Add 30 μL of diluted secondary antibody Anti-M13-HRP (1: 7000) , place at room temperature for 1 h, then wash the plate 6 times with PBST.
5) Stopping: Add 30 μL/well of TMB to develop color at room temperature for 5 min to 10 min, then add 30 μL/well of 2 M stop solution to stop the reaction. The OD values of each well was read at 450 nm with a microplate reader.
Results
Single domain antibodies having strong binding to the human BCMA protein were selected for further testing, including A016, A022, A026, A034, A043, A052, A058, A060, A068, A075, A077, A081, A095, A098, A119, A130, A134, A135 and A137. Their sequences are shown in Tables 1-2.
Table 1. Selected Antibody Sequences

The CDR sequences of these antibodies are summarized in Table 2.
Table 2. CDR Sequences


Example 2: Screening of Immunized Library
In this example, an immunized nanobody library was used to identify antibodies having satisfactory binding affinity and specificity. The top candidates and their sequences are shown in Tables 3 and 4.
Table 3. Top Antibodies from Immunized Library


Table 4. CDR sequences


Example 3: Binding of Single-Domain Library Antibodies to BCMA Expressing Cells
This example tested the antibodies for their binding affinity to the BCMA protein expressed on HEK cells and MM. 1S cells and BCMA negative HEK cells and CHO-S cells, as measured by flow cytometry (FACS) .
Cell plating
Cells were processed into cell suspension, and the density was adjusted to 1 × 106 cells/mL. The cell suspension was added to a 96-well round-bottom plate at 100 μL per well using a 100 μL pipette, followed by centrifugation at 300 g for 5 min to discard the supernatant.
Addition of antibody diluent
The antibodies to BCMA-HEK230 and MM. 1 S cells were diluted with FACS Buffer to 4 gradient concentrations of 150.00, 15.00, 1.50, and 0.15 nM. Antibodies to HEK293 cells were diluted with FACS Buffer into dilutions of 3 gradient concentrations of 300.00, 150.00 and 100.00 nM, antibodies to CHO-S cells were diluted with FACS Buffer into dilutions of 2 gradient concentrations of 300.00 and100.00 nM. The antibody dilution was added to a plate at 100 μL per well using a 100 μL pipette, and incubated at 4℃ for 60 min. Plate washing: The cells were washed twice with the FACS Buffer.
Addition of secondary antibody
The secondary antibody PE labeled anti-human IgG Fc was diluted in a ratio of 1: 300 using FACS Buffer, added to the corresponding wells at 100 μL/well, and incubated for 30 min at 4℃. The plate was washed twice with the FACS Buffer.
The results showed the FACS binding of the candidate antibodies to BCMA-HEK293, HEK293 and CHO-S cells, and A026 does not bind to BCMA-HEK293 (FIG. 1A-1B) . Non-specific binding test demonstrated that the candidates A052 exhibited non-specific binding to both HEK293 and CHO-S cells, while the others exhibited no non-specific binding (FIG. 2-3) . Candidates A034, A016, A026, A081, A043, A060, A077, A095 and A134 exhibited relatively weak binding to MM. 1S cells that expressed low levels of BCMA, while the other candidates exhibited no binding (FIG. 4A-4B) .
Example 4: Binding of Single-Domain Library Antibodies to Tumor Cells
This example tested the synthetic single-domain antibodies for their binding affinity to the tumor cells NCI-H929, U266 and OPM-2 cells, as measured by flow cytometry (FACS) .
Cell plating
Cells were processed into cell suspension, and the density was adjusted to 1 × 106 cells/mL. The cell suspension was added to a 96-well round-bottom plate at 100 μL per well using a 100 μL pipette, followed by centrifugation at 300 g for 5 min to discard the supernatant.
Addition of antibody diluent
The antibody was diluted with FACS Buffer to 8 gradient concentrations of 300.0000, 75.0000, 18.7500, 4.6875, 1.1719, 0.2930, 0.0732, and 0.0183 nM. The antibody diluent was added to a plate at 100 μL per well using a 100 μL pipette, and incubated at 4℃ for 60 min. Plate washing: The cells were washed twice with the FACS Buffer.
Addition of secondary antibody
The secondary antibody PE labeled anti-human IgG Fc was diluted in a ratio of 1: 300 using FACS Buffer, added to the corresponding wells at 100 μL/well, and incubated for 30 min at 4℃. The plate was washed twice with the FACS Buffer.
The results showed the FACS binding of the candidate antibodies to NCI-H929 cells (FIGs. 5A-5C and Table 5-1a-Table 5-1c) , U266 cells (FIGs. 6A-6C and Table 5-2a-Table 5-2c) and OPM-2 cells (FIGs. 7A-7C and Table 5-3a-Table 5-3c) . Candidate antibodies A016, A026, A034, A043, A060, A081, A098, A119, and A134 exhibited binding to U266 cells, while the others exhibited no binding (FIGs. 6A-6C and Table Table 5-2a-Table 5-2c) . Candidates A016, A022, A026, A034, A043, A060, A077, and A134 exhibited binding to OPM-2 cells (FIGs. 7A-7C and Table 5-3a-Table 5-3c) .
Table 5-1a. FACS binding of candidate antibodies to NCI-H929 cells

Table 5-1b. FACS binding of candidate antibodies to NCI-H929 cells

Table 5-1c. FACS binding of candidate antibodies to NCI-H929 cells

Table 5-2a. FACS binding of candidate antibodies to U266 cells

Table 5-2b. FACS binding of candidate antibodies to U266 cells


Table 5-2c. FACS binding of candidate antibodies to U266 cells

Table 5-3a. FACS binding of candidate antibodies to OPM-2 cells

Table 5-3b. FACS binding of candidate antibodies to OPM-2 cells

Table 5-3c. FACS binding of candidate antibodies to OPM-2 cells
As identified by the FACS, the binding of candidates to NCI-H929, U266, and OPM-2 tumor cells showed that 6 candidates, i.e., A016, A026, A034, A043, A060, and A134, exhibited binding to all three cell lines. None of the 6 candidate antibodies exhibited non-specific binding to HEK293 or CHO-S cells.
Nine molecules with signals on MM. 1S, NCI-H929, U266, and OPM-2 cells were selected, i.e., A016, A022, A034, A060, A068, A081, A098, A119, and A134 after excluding molecules with non-specific binding to HEK293 and CHO-S cells.
Example 5: Binding of Alpaca Immune Library Antibodies to Tumor Cells
This example tested the synthetic single-domain antibodies for their binding affinity to the tumor cells NCI-H929, U266 and OPM-2 cells, as measured by flow cytometry (FACS) .
Cell plating
Cells were processed into cell suspension, and the density was adjusted to 1 × 106 cells/mL. The cell suspension was added to a 96-well round-bottom plate at 100 μL per well using a 100 μL pipette, followed by centrifugation at 300 g for 5 min to discard the supernatant.
Addition of antibody diluent
The antibody was diluted with FACS Buffer to 8 gradient concentrations of 300.0000, 75.0000, 18.7500, 4.6875, 1.1719, 0.2930, 0.0732, and 0.0183 nM for NCI-H929 and U266 cells binding test. Antibodies to HEK293, CHO-S, and Jurkat cells were diluted with FACS Buffer into dilutions of 2 gradient concentrations of 300.0000 and 100.0000 nM. The antibody diluent was added to a plate at 100 μL per well using a 100 μL pipette, and incubated at 4℃ for 60 min. Plate washing: The cells were washed twice with the FACS Buffer.
Addition of secondary antibody
The secondary antibody PE labeled anti-human IgG Fc was diluted in a ratio of 1: 300 using FACS Buffer, added to the corresponding wells at 100 μL/well, and incubated for 30 min at 4℃. The plate was washed twice with the FACS Buffer.
FIGs. 8A-8E and Tables 6-1-6-5 show FACS binding of candidate antibodies to NCI-H929 cells. All candidate antibodies exhibited binding to the cells. Most of candidate antibodies exhibited superior binding to the cells compared to the positive antibody belantamab.
Table 6-1. FACS binding of candidate antibodies to NCI-H929 cells


Table 6-2. FACS binding of candidate antibodies to NCI-H929 cells

Table 6-3. FACS binding of candidate antibodies to NCI-H929 cells

Table 6-4. FACS binding of candidate antibodies to NCI-H929 cells

Table 6-5. FACS binding of candidate antibodies to NCI-H929 cells
FIGs. 9A-9E and Tables 7-1-7-5 show FACS binding of candidate antibodies to U266 cells. All candidate antibodies exhibited binding to the cells. Candidate antibodies all exhibited binding to the cells. Moreover, most of candidate antibodies exhibited superior binding to the cells comparable to the positive antibody belantamab.
Table 7-1. FACS binding of candidate antibodies to U266 cells


Table 7-2. FACS binding of candidate antibodies to U266 cells

Table 7-3. FACS binding of candidate antibodies to U266 cells

Table 7-4. FACS binding of candidate antibodies to U266 cells

Table 7-5. FACS binding of candidate antibodies to U266 cells
The FACS binding of candidate antibodies to HEK293 (FIGs. 10A-10B) , CHO-S (FIGs. 11A-11B) , and Jurkat cells (FIGs. 12A-12B) were also tested. The candidate antibodies B127 exhibited non-specific binding to CHO-S cell.
As identified by the FACS, the candidates exhibited binding to NCI-H929 and U266 tumor cells. Specifically, 8 candidate antibodies, i.e., B010, B030, B133, B136, B168, B172, B077, and B192, exhibited potent binding to both NCI-H929 and U266 cells. None of the 8 candidate antibodies exhibited non-specific binding to HEK293, CHO-S, or Jurkat cells.
17 molecules with signals on both NCI-H929 and U266 cells were selected, i.e., B255, B281, B171, B270, B282, B010, B030, B172, B231, B168, B127, B077, B192, B109, B136, B081, and B133 after excluding molecules with non-specific binding to HEK293, Jurkat, and CHO-S cells.
***
The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure covers the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims (24)

  1. A single domain antibody or a polypeptide comprising the single domain antibody, wherein the single domain antibody has binding specificity to the human B-cell maturation antigen (BCMA) protein and comprises a complementarity determining region 1 (CDR1) , a CDR2 and a CDR3, wherein the CDR1, CDR2, and CDR3, respectively, comprise the amino acid sequences of:
    SEQ ID NO: 95, 96 and 97;
    SEQ ID NO: 100, 101 and 102;
    SEQ ID NO: 105, 108 and 109; or
    SEQ ID NO: 103, 104 and 94.
  2. The antibody or polypeptide of claim 1, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 95, the CDR2 comprises the amino acid sequence of SEQ ID NO: 96, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 97.
  3. The antibody or polypeptide of claim 2, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 76.
  4. The antibody or polypeptide of claim 1, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 100, the CDR2 comprises the amino acid sequence of SEQ ID NO: 101, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 102.
  5. The antibody or polypeptide of claim 4, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 78.
  6. The antibody or polypeptide of claim 1, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 105, the CDR2 comprises the amino acid sequence of SEQ ID NO: 108, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 109.
  7. The antibody or polypeptide of claim 6, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 81.
  8. The antibody or polypeptide of claim 1, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 103, the CDR2 comprises the amino acid sequence of SEQ ID NO: 104, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 94.
  9. The antibody or polypeptide of claim 8, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 79.
  10. The antibody or polypeptide of any one of claims 1-9, wherein the polypeptide is a chimeric antigen receptor (CAR) or a bispecific antibody having a binding specificity to an antigen different from BCMA.
  11. A multispecific antibody comprising the antibody of any one of claims 1-9 and a second antibody or antigen-binding fragment having binding specificity to a second target antigen that is not BCMA.
  12. The multispecific antibody of claim 11, wherein the second target antigen is selected from the group consisting of GPRC5D, CD123, CS1, CD20, CD19, CD22, CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.
  13. A chimeric antigen receptor (CAR) comprising the antibody of any one of claims 1-9, a transmembrane domain, a costimulatory domain, and a CD3ξ intracellular domain.
  14. The CAR of claim 13, further comprises a second antibody or antigen-binding fragment having binding specificity to a second target antigen that is not BCMA.
  15. The CAR of claim 14, wherein the second target antigen is selected from the group consisting of GPRC5D, CD123, CS1, CD20, CD19, CD22, CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.
  16. A polynucleotide encoding the antibody or polypeptide of any one of claims 1-9 or the CAR of any one of claims 13-15.
  17. The polynucleotide of claim 16, which is one or more mRNA.
  18. The polynucleotide of claim 17, wherein the mRNA is chemically modified.
  19. A cell comprising the polynucleotide of any one of claims 16-18.
  20. An antibody-drug conjugate, comprising the antibody of any one of claims 1-9 conjugated to a drug moiety.
  21. A method of treating cancer in a patient in need thereof, comprising administering to the patient the antibody or polypeptide of any one of claims 1-9, the CAR of any one of claims 13-15, the polynucleotide of any one of claims 16-18, the cell of claim 19, or the antibody-drug conjugate of 20.
  22. The method of claim 21, wherein the cancer is a hematological cancer.
  23. The method of claim 22, wherein the hematological cancer is a BCMA-expressing B cell cancer.
  24. The method of claim 20, wherein the BCMA-expressing B cell cancer is multiple myeloma.
PCT/CN2024/140658 2023-12-20 2024-12-19 Single domain antibodies for bcma Pending WO2025130984A1 (en)

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