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WO2025216764A2 - Nanocorps anti-cd72 améliorés pour immunothérapie - Google Patents

Nanocorps anti-cd72 améliorés pour immunothérapie

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Publication number
WO2025216764A2
WO2025216764A2 PCT/US2024/054950 US2024054950W WO2025216764A2 WO 2025216764 A2 WO2025216764 A2 WO 2025216764A2 US 2024054950 W US2024054950 W US 2024054950W WO 2025216764 A2 WO2025216764 A2 WO 2025216764A2
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WO
WIPO (PCT)
Prior art keywords
car
cell
cells
nanobody
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/054950
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English (en)
Inventor
Arun WIITA
Matthew NIX
Adilia IZGUTDINA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California Berkeley
University of California San Diego UCSD
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Application filed by University of California Berkeley, University of California San Diego UCSD filed Critical University of California Berkeley
Publication of WO2025216764A2 publication Critical patent/WO2025216764A2/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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
    • C07K16/2851—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 lectin superfamily, e.g. CD23, CD72
    • 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

Definitions

  • Anti-CD72 nanobodies have been developed as therapeutic agents for the treatment of malignancies that express CD72, including B-cell malignancies (see, e.g., W02021/003428 and WO2022/150389). Such nanobodies can be used, for example, in immunotherapies such as chimeric T-cell receptor-based therapies that target CD72.
  • the present disclosure features an affinity matured anti-CD72 antibody that confers more robust anti-tumor activity compared to closely related sequences, particularly in patients that have cancer cells that have low CD72 antigen density. Accordingly, the present disclosure provides a nanobody comprising a VH region that specifically binds to CD72, wherein the nanobody comprises a CDR1 sequence comprising TISPIDQ (SEQ ID NO:2), a CDR2 sequence comprising FVAAIFLGGN (SEQ ID NO:3), and a CDR3 sequence comprising VGYVDKWDDSNYHT (SEQ ID NO:4).
  • the nanobody comprises a framework comprising a framework region having at least 90% or at least 95% identity to a framework comprising an FR1 sequence EVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 5), an FR2 sequence MSWYRQAPGKERE (SEQ ID NO: 6), an FR3 sequence TYYADSVKGRFTISRDNSQNTLYLQMNSLRAEDTAVYYCA (SEQ ID N0:7), and an FR4 sequence YWGQGTQVTVSS (SEQ ID N0:8).
  • a nanobody that specifically binds to CD72 comprises: a CDR1 sequence comprising TISPIDQ, a CDR2 sequence comprising FVAAIFLGGN, a CDR3 sequence comprising VGYVDKWDDSNYHT; and comprises an FR1, an FR2, an FR3, and an FR4 as follows: the FR1 comprises SEQ ID NO:9 or SEQ ID NO:5; the FR2 comprises SEQ ID NO: 10 or SEQ ID NO:6; the FR3 comprises SEQ ID NO: 11 or comprises one or more of the following: S at position 74, L at position 78, or R at position 86; and the FR4 comprises SEQ ID NO:8.
  • the FR3 comprises SEQ ID NO:7.
  • the nanobody comprises one or more of the following: FR1 comprises E at position 1; the FR2 comprises S at position 35; the FR3 compirses S at position 74 and L at position 78, or S at position 74 and R at position 86, or L at position 78 and R at position 86, or S at position 74, L at position 78, and R at position 86.
  • the nanobody comprises SEQ ID NO: 1.
  • a method of treating a hematological malignancy that comprises malignant B cells that express CD72 or a malignancy that comprises malignant myeloid cells that express CD72 comprising administering a plurality of immune effector cells genetically modified to express one or more anti-CD72 nanobodies as described herein to a subject that has the hematological malignancy.
  • the hematological malignancy is a B-cell leukemia, e.g., chronic lynmphocytic leukemia.
  • the hematological malignancy is a non-Hodgkin’s lymphoma. In some embodiments, the hematological malignancy is multiple myeloma. In some embodiments, the tumor cells have a low density of CD72.
  • a method of treating an autoimmune disease comprising administering a plurality of immune effector cells genetically modified to express one or more anti-CD72 nanobodies as described herein to a subject that has the autoimmune.
  • the disclosure provides a polynucleotide encoding an anti-CD72 nanobody as described here.
  • the disclosure provides a polynucleotide encoding a CAR comprising one or more anti-CD72 nanobody of the present invention.
  • the disclosure provides vectors comprising such polynucleotides and mammalian host cells, e.g., immune effector cells, comprising the polynucleotides.
  • the vector a retroviral vector, e.g., a self-inactivating lentiviral vector.
  • the immune effector cell is a T lymphocyte or NK cell.
  • FIG. 1 DNA sequences of nanobody-based CARs that bind CD72.
  • AR binders Alignment of DNA sequence of CD72 CAR binders: NbD4, humanized H24, affinity matured NbD4.13, and humanized affinity matured NbD4.13.
  • FIG. 3A-B CAR-T cells generated with affinity -matured CD72 nanobodies demonstrate superior anti-tumor potency to H24.
  • FIG. 4A-B CAR-T cells generated with affinity -matured CD72 nanobodies demonstrate superior killing of JeKo-CD72 antigen low cell line.
  • FIG. 5A-D Affinity matured NbD4.13 significantly prolongs survival in vivo compared to H24 in mantle cell lymphoma model.
  • C) Kaplan- Meier survival curves of mice treated with empty CAR, H24 and NbD4.13 CAR. P values obtained with the log-rank (Mantel-Cox) test * p ⁇ 0.05.
  • FIG. 6A-B Affinity matured NbD4.13 CAR outperformed H24 in a cytotoxicity assay against ex vivo JeKo-1 CD72 “low” post-relapsed mouse tumor cells.
  • FIG. 7A-C Affinity matured CAR-T NbD4.13 showed greater cytokine secretion in response to JeKo-1 CD72 antigen low cell line and faster tumor clearance in vitro.
  • FIG. 8A-D Affinity matured, humanized CAR conferred a survival benefit versus JeKo-1 -CD72 antigen low model.
  • B-cell differentiation antigen CD72 or “CD72” (also referred to as lyb-2) is used herein to refer to a polypeptide that is encoded by a CD72 gene cytogenetically localized to human chromosome 9pl3.3 (genomic coordinates (GRCh38/hg38 assembly December 2013: 9:35,609,978-35,618,426) and plays a role in B-cell proliferation and differentiation.
  • a human CD72 protein sequence encoded by the CD72 gene is available under Uniprot accession number P21854.
  • CD72 is a single-pass Type-II membrane protein with an extracellular C-type lectin domain and cytoplasmic ITIM motifs.
  • CD72 has been shown to interact with the B-cell receptor complex and play a role in the normal function of B-cell signaling. It is similar to the CD22 receptor which also possesses cytoplasmic ITIM motifs. The ITIM motifs of CD72 and CD22 both function to bind to SHP-1, a protein that can interact with members of the BCR signaling chain and suppress BCR signaling as part of shaping B-cell immune tolerance. Genetic ablation of CD72 in mice was not lethal, but such mice exhibited increased immune system activation, lending evidence to its roles as a BCR inhibitory molecule. CD72 therefore is considered to be an inhibitory receptor for BCR signaling.
  • nanobody refers to a single-domain antibody comprising a single monomeric variable antibody domain that can form a functional antigen binding site without interaction with another variable domain, e.g., without a VH/VL interaction as is required between the VH and VL domains of a conventional 4-chain monoclonal antibody).
  • a nanobody of the present invention can be incorporated into antibodies having various formats, including, e.g., a bivalent or multivalent antibody format that comprises other antibody binding domains, which may have the same, or a different, binding specificity.
  • a nanobody of the present invention may thus be part of a larger molecule such as a multivalent or multispecific immunoglobulin that includes more than one moiety, domain or unit.
  • a nanobody may also be part of a larger molecule that comprises another functional element, such as, for example, a half-life extender (HLE), targeting unit and/or a small molecule such a polyethyleneglycol (PEG).
  • HLE half-life extender
  • PEG polyethyleneglycol
  • the term “nanobody” includes humanized versions of nanobodies.
  • V-region refers to an antibody, e.g., nanobody, variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4, which segments are added to the V- segment as a consequence of rearrangement of V-region genes during B-cell differentiation.
  • CDR complementarity-determining region
  • HVR hypervariable regions
  • the amino acid sequences of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J.Mol.Biol 1997, 273(4)).
  • IMGT ImMunoGeneTics database
  • antigen combining sites are also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc,M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan l;29(l):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci.
  • Epitopes refers to a site on an antigen to which an antibody binds.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).
  • valency refers to the number of different binding sites of an antibody for an antigen.
  • a monovalent antibody comprises one binding site for an antigen.
  • a multivalent antibody comprises multiple binding sites.
  • identity in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region. Alignment for purposes of determining percent amino acid sequence identity can be performed in various methods, including those using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • BLAST 2.0 can be used with the default parameters to determine percent sequence identity.
  • amino acid residue in a variable domain polypeptide refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence.
  • an amino acid residue in a variable domain polypeptide “corresponds to” an amino acid in the variable domain polypeptide of SEQ ID NO: 1 when the residue aligns with the amino acid in SEQ ID NO: 1 when optimally aligned to SEQ ID NO: 1.
  • the polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.
  • a “conservative” substitution as used herein refers to a substitution of an amino acid such that charge, hydrophobicity, and/or size of the side group chain is maintained.
  • Illustrative sets of amino acids that may be substituted for one another include (i) positively- charged amino acids Lys, Arg and His; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) large aliphatic nonpolar amino acids Vai, Leu and He; (vi) slightly polar amino acids Met and Cys; (vii) small-side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gin and Pro; (viii) aliphatic amino acids Vai, Leu, He, Met and Cys; and (ix) small hydroxyl amino acids Ser and Thr.
  • Reference to the charge of an amino acid in this paragraph refers to the charge at physiological pH.
  • nucleic acid and “polynucleotide” are used interchangeably and as used herein refer to both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above.
  • a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide, and combinations thereof.
  • the terms also include, but is not limited to, single- and doublestranded forms of DNA.
  • a polynucleotide e.g., a cDNA or mRNA
  • a polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
  • the nucleic acid molecules may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analogue, intemucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • a reference to a nucleic acid sequence encompasses its complement unless otherwise specified.
  • a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.
  • the term also includes codon- optimized nucleic acids that encode the same polypeptide sequence.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • a “vector” as used here refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".
  • the terms “subject”, “patient” or “individual” are used herein interchangeably to refer to any mammal, including, but not limited to, a human.
  • the animal subject may be, a primate (e.g., a monkey, chimpanzee), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig), or any other mammal.
  • the subject”, “patient” or “individual” is a human.
  • anti-CD72 binding domains that can be used for diagnostic and therapeutic purposes.
  • an anti-CD72 nanobody of the present disclosure has a KD of less than 5 nM or less than 1 nM, e.g., as measured by Bio-layer interferometry as illustrated in the Technical Section of the application.
  • an anti-CD72 nanobody featured in the present disclosure has three CDRs of a variable domain sequence of SEQ ID NO: 1 and comprises a framework region in which FR1 has at least 90% identity to SEQ ID NO:5, FR2 has at leat 90% identity to SEQ ID NO: 6, FR3 has at least 90% identity to SEQ ID NO: 7 and FR4 has at least 90% identity to SEQ ID NO:8 and comprises at least one of the following: FR1 comprises E at position 1; FR2 comprises S at position 35; and FR3 comprises S at position 74, L at position 78, or R at position 86.
  • an anti-CD72 nanobody of the present invention comprises a CDR1 comprising SEQ ID NO:2, a CDR2 comprising SEQ ID NO:3, and a CDR3 comprising SEQ ID NO:4, and has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of each of the FR regions of the variable region sequence of SEQ ID NO: 1.
  • the variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in SEQ ID NO: 1.
  • a nanobody of the present invention comprises an FR1-FR2-FR3-FR4 framework sequence that has at least 95% identity to the FR1-FR2-FR3-FR4 framework sequence of SEQ ID NO: 1.
  • FR1-FR2-FR3- FR4 is intended to refer to the framework sequence across its length, i.e., the sequence of SEQ ID NO: 1 from the N-terminus to the C-terminus without the three CDR sequences.
  • the FR1 region of a nanobody of the present invention comprises an FR1 sequence having at least 90% identity, or at least 95% identity to the FR1 sequence of SEQ ID NO: 5.
  • the FR2 region of a nanobody of the present invention comprises an FR2 sequence having at least 90% identity, or at least 95% identity to the FR2 sequence of of SEQ ID NO:6.
  • the FR3 region of a nanobody of the present invention comprises an FR3 sequence having at least 90% identity, or at least 95% identity to the FR3 sequence of SEQ ID NO:7.
  • the FR4 region of a nanobody of the present invention comprises an FR4 sequence having at least 90% identity, or at least 95% identity to the FR4 sequence of of SEQ ID NO: 8.
  • an anti-CD72 nanobody of the present invention comprises a CDR1 comprising SEQ ID NO:2, a CDR2 comprising SEQ ID NO:3, and a CDR3 comprising SEQ ID NON; an FR1 comprising SEQ ID NO:9, or variant thereof comprising E at position 1; an FR2 comprising SEQ ID NO: 10, or variant thereof comprising S at position 35; an FR3 comprising SEQ ID NO: 11 or a variant thereof comprising one or more of the following: S at position 74, L at position 78, or R at position 86; and an FR4 comprising SEQ ID NO:8.
  • the FR1 comprises E at position 1; the FR2 comprises S at position 35; the FR3 compirses S at position 74 and L at position 78, or S at position 74 and R at position 86, or L at position 78 and R at position 86, or S at position 74, L at position 78, and R at position 86.
  • the nanobody comprises SEQ ID NO: 1.
  • a nanobody of the present invention may be incorporated into a bivalent antibody or a multivalent antibody that binds to the same, or a different, antigen.
  • a nanobody of the present invention may be incorporated into a bispecific antibody or multispecific antibody that binds to an antigen at different epitopes, or that binds to different antigens.
  • such an antibody may comprise an Fc region.
  • a nanobody of the present invention may be present as an antigen binding domain of a larger molecule, e.g., present as an antigen binding domain of a chimeric antigen receptor or synthetic Notch receptor, as further detailed below.
  • a bispecific antibody, multispecific antibody, chimeric antibody receptor, synthetic Notch receptor, or other nanobody-containing construct may comprise more than one anti-CD72 nanobodies, e.g., two, three, or four anti-CD72 nanobodies, e.g., where the nanobodies are joined by linkers.
  • a nanobody of the present disclosure is linked to a second nanobody, e.g., a second anti-CD72 nanobody, or to an scFV antibody to form a bi-specific antibody.
  • an anti-CD72 nanobody of the present invention may be incorporated into a bispecific antibody having a second binding domain that targets an antigen on an immune effector cell, such as a T cell.
  • a bispecific antibody may comprise an anti-CD72 nanobody of the present disclosure and an antibody, e.g., scFv, that targets CD3 or an anti-CD16 scFv for engaging NK cells.
  • a bispecific antibody comprises an anti-CD72 nanobody as described herein and an antibody, e.g., scFV, that targets CD28.
  • any of the nanobodies or antibodies described herein are linked to a toxin or drug, e.g., to form an antibody-drug conjugate (ADC) .
  • the nanobody is attached to a therapeutic cytotoxic/cytostatic drug.
  • the drug of the ADC include, but are not limited to, microtubule inhibitors and DNA-damaging agents, polymerase inhibitors (e.g. , the polymerase II inhibitor, a-amanitin), and the like.
  • the antibody is conjugated to the drag directly or through a linker, while in other embodiments, the antibody is conj gated to a drug carrier (e.g., a liposome containing the drug, a polymeric drug carrier, a nanoparticle drug carrier, a lipid drug carrier, a dendrimeric drag carrier, and the like).
  • a drug carrier e.g., a liposome containing the drug, a polymeric drug carrier, a nanoparticle drug carrier, a lipid drug carrier, a dendrimeric drag carrier, and the like.
  • Chimeric antigen receptors are recombinant receptor constructs comprising an extracellular antigen-binding domain (e.g., a nanobody) joined to a transmembrane domain, and further linked to an intracellular signaling domain (e.g., an intracellular T cell signaling domain of a T cell receptor) that transduces a signal to elicit a function.
  • an intracellular signaling domain e.g., an intracellular T cell signaling domain of a T cell receptor
  • immune cells e.g., T cells or natural killer (NK) cells
  • NK cells natural killer cells
  • effector cells e.g., cytotoxic and/or memory functions of T cells or NK cells.
  • the components include an extracellular targeting domain, a transmembrane domain and intracellular signaling/activation domain, which are typically linearly constructed as a single fusion protein.
  • the extracellular region comprises an anti-CD72 nanobody as described herein.
  • the "transmembrane domain” is the portion of the CAR that links the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the host cell that is modified to express the CAR, e.g., the plasma membrane of an immune effector cell.
  • the intracellular region may contain a signaling domain of TCR complex, and/or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137) and OX-40 (CD 134).
  • a "first-generation CAR” generally has a CD3-zeta signaling domain.
  • Additional costimulatory intracellular domains may also be introduced (e.g., second and third generation CARS) and further domains including homing and suicide domains may be included in CAR constructs.
  • CAR components are further described below.
  • a chimeric antigen receptor of the present disclosure comprises an extracellular antigen-binding domain that comprises an anti-CD72 nanobody domain having a CDR1, CDR2, and CDR3 as described herein.
  • the anti-CD72 nanobody domain comprises a humanized version of SEQ ID NO: 12, in which residues in the framework are substituted to provide a framework sequence FR1-FR2-FR3-FR4 that has at least 85%, or at least 90%, or at least 95%, or greater, to a human VH framework, e.g., a human germline framework, FR1-FR2-FR3-FR4.
  • the extracellular domain may comprise two more anti-CD72 nanobodies.
  • the extracellular domain may comprise three of four different nanobodies that are described herein.
  • the extracellular domain may comprises multiple copies of the same nanobody.
  • the extracellular domain may comprise a nanobody as described herein and an anti-CD72 nanobody that binds to a different CD72 epitope.
  • at least one of the nanobodies comprises a CDR1 sequence comprising TISPIDQ, a CDR2 sequence comprising FVAAIALGGN or and a CDR3 sequence comprising VGYVDKWDDSNYHT.
  • a CAR construct encoding a CAR may also comprise a sequence that encodes a signal peptide to target the extracellular domain to the cell surface.
  • the CAR may one or more hinge domains that link the antigen binding domain comprising an anti-CD72 nanobody of the present invention and the transmembrane domain for positioning the antigen binding domain.
  • a hinge domain may be derived either from a natural, synthetic, semi -synthetic, or recombinant source.
  • the hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region, e.g., a naturally occurring human immunglobulin hinge region, or an altered immunoglobulin hinge region.
  • Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, PD1, CD 152, and CD7, which may be wild-type hinge regions from these molecules or may be altered.
  • transmembrane suitable for use in a CAR construct may be employed.
  • Such transmembrane domains include, but are not limited to, all or part of the transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD I la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB 1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNA
  • a transmembrane domain incorporated into a CAR construct may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • a CAR construct of the present disclosure includes one or more intracellular signaling domains, also referred to herein as co-stimulatory domains, or cytoplasmic domains that activate or otherwise modulate an immune cell, (e.g., a T lymphocyte, NK cell, iNKT cell, macrophage, gamma delta T cell).
  • the intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced.
  • a co-stimulatory domain is used that increases CAR immune T cell cytokine production.
  • a co- stimulatory domain is used that facilitates immune cell (e.g., T cell) replication.
  • a co-stimulatory domain is used that prevents CAR immune cell (e.g., T cell) exhaustion.
  • a co-stimulatory domain is used that increases immune cell (e.g., T cell) antitumor activity.
  • a co-stimulatory domain is used that enhances survival of CAR immune cells (e.g., T cells) (e.g., post-infusion into patients).
  • intracellular signaling domains for use in a CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
  • TCR T cell receptor
  • co-receptors that act in concert to initiate signal transduction following antigen receptor engagement
  • a primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • Examples of IT AM containing primary intracellular signaling domains include those of CD3 zeta, common FcR gamma, Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.
  • a CAR comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3- zeta.
  • An intracellular signaling domain of a CAR can comprise a primary intracellular signaling domain only, or may comprise additional desired intracellular signaling domain(s) useful in the context of a CAR of the invention.
  • the intracellular signaling domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling domain.
  • the costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen.
  • LFA-1 lymphocyte function-associated antigen-1
  • CD2 CD7
  • LIGHT NKG2C
  • B7-H3 B7-H3
  • ligand that binds to CD83 and the like.
  • CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3):696-706).
  • costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB 1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM,
  • a CAR may be designed to exhibit conditional expression, e.g., designed as an inducible CAR, or may otherwise comprise a mechanism for reversibly expressing the CAR, or controlling CAR activity to largely restrict it to a desired environment.
  • the CAR may comprise a safety switch gene.
  • the CAR may be an on-switch CAR or an off-switch CAR (see, e.g., Jan et al, Sci Transl Med. Jan 6: 13(575):eabb6295, 2021).
  • the CAR-expressing cell uses a split CAR.
  • a split CAR system comprises a cell expressing a first CAR having a first antigen binding domain and a costimulatory domain (e.g., 4 IBB), and the cell also expresses a second CAR having a second antigen binding domain and an intracellular signaling domain (e.g., CD3 zeta).
  • a costimulatory domain e.g. 4 IBB
  • an intracellular signaling domain e.g., CD3 zeta
  • a host cell e.g., a T cell
  • a synthetic Notch receptor comprising an extracellular domain that targets one antigen induces the expression of a CAR that targets a second antigen.
  • a synNotch comprises a one or more anti-CD72 nanobodies as described herein.
  • one or more anti-CD72 nanobodies is incorporated into a CAR, the expression of which is activated by a synNotch expressed by the host cell.
  • a cell expressing a CAR comprising one or more anti-CD72 nanobodies expresses a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g., that binds to the same target or a different target (e.g., a target other than CD72, e.g., CD22 or CD 19, that is expressed on a B cell malignancy.
  • a second CAR e.g., a second CAR that includes a different antigen binding domain, e.g., that binds to the same target or a different target (e.g., a target other than CD72, e.g., CD22 or CD 19, that is expressed on a B cell malignancy.
  • a cell expressing an anti-CD72 nanobdy comprises an alternative chimeric antigen receptor, such as an HLA-Independent TCR-based Chimeric Antigen Receptor (also known as “HIT-CAR”, e.g., those disclosed in International Patent Application No. PCT/US19/017525), T cell receptor fusion constructs (TRuCs) (e.g., those disclosed in Baeuerle et al., “Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response,” Nature Comm. 10: 2087 (2019), synthetic T cell receptor and antigen receptors (STARs) (e.g., those disclosed in Liu et al.
  • HIT-CAR also known as “HIT-CAR”
  • T cell receptor fusion constructs TRuCs
  • TRuCs synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response
  • STARs synthetic T cell receptor and antigen receptors
  • the invention is not limited by the type of immune cells genetically modified to express a CAR, or synthetic Notch receptor.
  • Illustrative immune cells include, but are not limited to, T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, macrophages, and myeloid-derived phagocytes.
  • T cells that can be modified to express CARs include memory T cells, CD4+, and CD8+ T cells.
  • the immune cells e.g., T cells, are autologous cells from the patient to undergo immunotherapy.
  • the immune cells are allogeneic.
  • Immune effector cells such as T cells may be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 2006/0121005.
  • immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • T cells e.g., alpha/beta T cells and gamma/delta T cells
  • B cells natural killer (NK) cells
  • natural killer T (NKT) cells e.g., myeloid-derived phagocytes.
  • Any method may be used to genetically modify an effector cells, such as a T-cell or NK cell to express a CAR comprising an anti-CD72 nanobody of the present invention.
  • methods of genetically engineering immune cells include, but are not limited to, retrovirus- or lentivirus-mediated transduction.
  • Other viral delivery systems include adenovirus, adeno-associated virus, herpes simplex viral vectors, pox viral vectors, alphavirus vectors, poliovirus vectors, and other positive and negative stranded RNA viruses, viroids, and virusoids, or portions thereof.
  • Methods of transduction include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al. Blood 80: 1418-1422 (1992), or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al. Exp. Hemat.
  • genetic modification is performed using transposase-based systems for gene integration, CRISPR/Cas-mediated gene integration, TALENS or Zinc- finger nucleases integration techniques.
  • CRISPR/Cas-mediated gene integration may be employed to introduce a CAR, or synthetic Notch receptor, or other synthetic chimeric TCR-like receptors into immune effectors cells, which may then be selected and expanded for administration to a patient.
  • an anti-CD72 nanbody of the present disclosure may be conjugated or linked, either directly or indirectly, to therapeutic and/or imaging/detectable moieties.
  • a nanobody or the present invention, or an antigen binding region comprising a nanobody of the present invention may be conjugated to agents including, but not limited to, a detectable marker, a cytotoxic agent, an imaging agent, a therapeutic agent, or an oligonucleotide.
  • agents including, but not limited to, a detectable marker, a cytotoxic agent, an imaging agent, a therapeutic agent, or an oligonucleotide.
  • an anti-CD72 nanobody of the present invention is conjugated to cytotoxic moiety or other moiety that inhibits cell proliferation.
  • the antibody is conjugated to a cytotoxic agent including, but not limited to, e.g., ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, methotrexact, actinomycin, a diphtheria toxin, extotoxin A from Pseudomonas, Pseudomonas exotoxin40, abrin, abrin A chain, modeccin A chain, alpha sarcin, gelonin, mitogellin, restrict
  • the antibody may be linked to an agent such as an enzyme inhibitor, a proliferation inhibitor, a lytic agent, a DNA or RNA synthesis inhibitors, a membrane permeability modifier, a DNA metabolite, a dichloroethylsulfide derivative, a protein production inhibitor, a ribosome inhibitor, or an inducer of apoptosis.
  • an anti-CD72 nanobody of the present invention, or an antigen binding domain comprising an anti-CD72 nanobody of the present invention may be linked to a radionuclide, an iron-related compound, a dye, a fluorescent agent, or an imaging agent.
  • an antibody may be linked to agents, such as, but not limited to, metals; metal chelators; lanthanides; lanthanide chelators; radiometals; radiometal chelators; positron-emitting nuclei; microbubbles (for ultrasound); liposomes; molecules microencapsulated in liposomes or nanosphere; monocrystalline iron oxide nanocompounds; magnetic resonance imaging contrast agents; light absorbing, reflecting and/or scattering agents; colloidal particles; fluorophores, such as near-infrared fluorophores.
  • agents such as, but not limited to, metals; metal chelators; lanthanides; lanthanide chelators; radiometals; radiometal chelators; positron-emitting nuclei; microbubbles (for ultrasound); liposomes; molecules microencapsulated in liposomes or nanosphere; monocrystalline iron oxide nanocompounds; magnetic resonance imaging contrast agents; light absorbing, reflecting and/or scattering agents; colloidal particles; fluorophores, such as
  • An anti-CD72 nanobody, an antigen binding molecule comprising an anti-CD72 nanobody, or an effector cell, e.g., T-cell, genetically modified to express a CAR or alternative chimeric receptor as described herein comprising an anti-CD72 nanobody of the present invention can be combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), and cells transfected with genes encoding immune stimulating cytokines (He et al. (2004) J. Immunol. 173:4919-28).
  • an immunogenic agent such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), and cells transfected with genes encoding immune stimulating cytokines (He et al. (2004) J. Immunol. 173:4919-28).
  • Non-limiting examples of cancer vaccines that can be used include t cells transfected to express the cytokine GM-CSF, DNA-based vaccines, RNA- based vaccines, and viral transduction-based vaccines.
  • the cancer vaccine may be prophylactic or therapeutic.
  • an anti-CD72 nanobody as described herein, an antigen binding molecule comprising an anti-CD72 nanobody, or an effector cell, e.g., T-cell, genetically modified to express a CAR or alternative chimeric receptor as described herein comprising an anti-CD72 nanobody of the present invention is co-administered with an immunomodulating agent.
  • immodulating agents include, but are not limited to, cytokines, growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-15, an IL-15/IL- 15Ra, e.g., sushi domain, complex, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF), interferons (e.g., interferon-a, -0 or -y), erythropoietin and thrombopoietin, or a combination thereof.
  • cytokines interleukin-1
  • IL-2 interleukin-2
  • IL-3 IL-6
  • IL-10 IL-12
  • IL-15
  • the complex may be coadministered with an adjuvant, such as a Toll-like receptor (TLR) agonist, a C-type lectin receptor (CLR) agonist, a retinoic acid-inducible gene I-like receptor (RLR) agonist, a saponin, a polysaccharide such as chitin, chitosan, 0-glucan, an ISCOM, QS-21, or another immunopotentiating agent.
  • TLR Toll-like receptor
  • CLR C-type lectin receptor
  • RLR retinoic acid-inducible gene I-like receptor
  • An anti-CD72 nanobody of the present disclsoure including embodiments in which the anti-CD72 nanobody is provided as a component of an antigen binding molecule, such as a bivalent or multivalent antibody, or is provided as a component of a CAR molecule, or other therapeutic chimeric receptor described herein, can be used to treat any malignancy that expresses CD72.
  • the malignancy is a B cell malignancy.
  • B-cell malignancies include, but are not limited to, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia/small lymphocytic lymphoma, monoclonal B-cell lymphocytosis, B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic B-cell lymphoma/leukemia, unclassifiable, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, monoclonal gammopathy of undetermined significance (MGUS) IgM, p heavy-chain disease, y heavy-chain disease, a heavy-chain disease, MGUS IgG/A, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, monoclonal immunoglobulin deposition diseases, extranod
  • a malignancy treated with an anti-CD72 nanobody as described herein is Hodgkin lymphoma, e.g., nodular lymphocyte predominant Hodgkin lymphoma, or classical Hodgkin lymphoma, including nodular sclerosis classical Hodgkin lymphoma, lymphocyte-rich classical Hodgkin lymphoma, mixed cellularity classical Hodgkin lymphoma, and lymphocyte-depleted classical Hodgkin lymphoma.
  • Hodgkin lymphoma e.g., nodular lymphocyte predominant Hodgkin lymphoma, or classical Hodgkin lymphoma, including nodular sclerosis classical Hodgkin lymphoma, lymphocyte-rich classical Hodgkin lymphoma, mixed cellularity classical Hodgkin lymphoma, and lymphocyte-depleted classical Hodgkin lymphoma.
  • a malignancy treated with an anti-CD72 nanobody as described herein in a posttransplant lymphoproliferative disorder such as plasmacytic hyperplasia PTLD, infectious mononucleosis PTLD, florid follicular hyperplasia PTLD, polymorphic PTLD, monomorphic PTLD (B- and T-/NK-cell types), or classical Hodgkin lymphoma PTLD.
  • a malignancy treated with an anti-CD72 nanobody therapeutic agent as described herein is T-cell acute lymphoblastic leukemia, acute myeloid leukemia, or T-cell acute lymphoblastic leukemia.
  • a therapeutic agent comprising an anti-CD72 nanobody as described herein is used to treat a patient that has cancer cells, e.g., lymphomas cells, that have a low density of CD72.
  • cancer cells e.g., lymphomas cells
  • a “low density” of CD72 for purposes of this disclosure is considered to be less than 10,000 CD72 protein copies at the plasma membrane per cell.
  • An anti-CD72 nanobody of the present disclosure including embodiments in which the anti-CD72 nanobody is provided as a component of an antigen binding molecule, such as a bivalent or multivalent antibody, or is provided as a component of a CAR molecule, or other therapeutic chimeric receptor described herein, can be used to treat an autoimmune disease in a subject.
  • an antigen binding molecule such as a bivalent or multivalent antibody
  • CAR molecule or other therapeutic chimeric receptor described herein
  • autoimmune diseases include, but are not limited to, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, cicatricial pemphigoid, autoimmune alopecia, Graves’ disease, Hashimoto’s thyroiditis, autoimmune haemolytic anaemia, cryoglobulinemia, pernicious anaemia, myasthenia gravis, neuromyelitis optica, autoimmune epilepsy, encephalitis, autoimmune hepatitis, chronic autoimmune urticaria, linear IgA disease, IgA nephropathy, vitiligo, primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune thrombocytopenic purpura, autoimmune Addison’s disease, multiple sclerosis, Type 1 diabetes mellitus, dermatitis herpetiformis, coeliac disease, psoriasis, dermatomyositis, polymy
  • a method of treating a B-cell malignancy or autoimmune disease using an anti-CD72 nanobody or antigen binding molecule, e.g., an antibody, that comprises the anti-CD72 nanobody comprises administering the anti-CD72 nanobody or antigen binding molecule that comprises the anti-CD72 nanobody as a pharmaceutical composition to a patient in a therapeutically effective amount using a dosing regimen suitable for treatment of the B-cell malignancy or autoimmune disease.
  • the composition can be formulated for use in a variety of drug delivery systems.
  • One or more physiologically acceptable excipients or carriers can also be included in the compositions for proper formulation. Suitable formulations for use in the present invention are found, e.g., in Remington: The Science and Practice of Pharmacy, 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, 2005.
  • the nanobody (or antibody or antigen binding molecule comprising the nanobody) is provided in a solution suitable for administration to the patient, such as a sterile isotonic aqueous solution for injection.
  • the antibody is dissolved or suspended at a suitable concentration in an acceptable carrier.
  • the carrier is aqueous, e.g., water, saline, phosphate buffered saline, and the like.
  • the compositions may contain auxiliary pharmaceutical substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and the like.
  • the pharmaceutical compositions are administered to a patient in an amount sufficient to cure or at least partially arrest the disease or symptoms of the disease and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
  • a therapeutically effective dose is determined by monitoring a patient’s response to therapy. Typical benchmarks indicative of a therapeutically effective dose include the amelioration of symptoms of the disease in the patient. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health, including other factors such as age, weight, gender, administration route, etc. Single or multiple administrations of the antibody may be administered depending on the dosage and frequency as required and tolerated by the patient.
  • the methods provide a sufficient quantity of anti-CD72 nanobody or antigen binding molecule that comprises the anti-CD72 nanobody to effectively treat the patient.
  • the nanobody can be administered by any suitable means, including, for example, parenteral, intrapulmonary, and intranasal administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the nanobody may be administered by insufflation.
  • the nanobody may be stored at 10 mg/ml in sterile isotonic aqueous saline solution for injection at 4°C and is diluted in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to the patient.
  • the nanobody is administered by intravenous infusion over the course of 1 hour at a dose of between 0.01 and 25 mg/kg.
  • the nanobody is administered by intravenous infusion over a period of between 15 minutes and 2 hours.
  • the administration procedure is via sub-cutaneous bolus injection.
  • the dose of nanobody is chosen in order to provide effective therapy for the patient and is in the range of less than 0.01 mg/kg body weight to about 25 mg/kg body weight or in the range 1 mg - 2 g per patient.
  • the dose is in the range 0.1 - 10 mg/kg or approximately 50 mg - 1000 mg / patient.
  • the dose may be repeated at an appropriate frequency which may be in the range once per day to once every three months, or every six months, depending on the pharmacokinetics of the nanobody (e.g., half-life of the antibody in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect of the antibody).
  • the in vivo half-life of between about 7 and about 25 days and antibody dosing is repeated between once per week and once every 3 months or once every 6 months.
  • the nanobody is administered approximately once per month.
  • compositions of the present invention comprise CAR-expressing immune effector cells e.g., a plurality of CAR-expressing immune effector cells that are genetically modified to express a CAR comprising an anti-CD72 nanobody as described herein.
  • Such cells may be formulated with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients, e.g., buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • diluents or excipients e.g., buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids
  • antioxidants e.g., antioxidants
  • chelating agents such as EDTA or glutathione
  • adjuvants e.g
  • compositions comprising the CAR-modified immune effector cells may be administered in a manner appropriate to the B-cell malignancy to be treated.
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
  • a pharmaceutical composition comprising CAR-modified immune effector cells, e.g., T cells or NK cells, as described herein are administered at a dosage of 10 4 to 10 9 cells/kg body weight, in some instances 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges.
  • the cells e.g., T cells or NK cells modified as described herein, may be administered at 3 x 10 4 , l x 10 6 , 3 x 10 6 , or 1 x 10 7 cells/kg body weight.
  • the cell compositions may also be administered multiple times at these dosages.
  • the genetically modified immune effector cells are administered intravenously.
  • Such cells are administered to a patient by intradermal or subcutaneous injection.
  • the CAR-expressing cells may also be injected directly into a particular site, such as a lymph node.
  • a subject may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T or NK cells.
  • leukocytes e.g., T cell or NK cell isolates
  • These cell isolates e.g., T cell or NK cell isolates, may be expanded by methods known in the art and treated such that one or more CAR constructs of the invention may be introduced, thereby creating a CAR-expressing cell, e.g., CAR-T cell or CAR-expressing NK cell, of the invention.
  • Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of the expanded CAR-expressing cells of the present invention.
  • expanded cells are administered before or following surgery.
  • lymphodepletion e.g., using melphalan, cytoxan, cyclophosphamide, or fludarabind, is performed on a subject, e.g., prior to administering a population of immune effectors cells that express a CAR comprising an anti-CD72 nanobody of the present invention.
  • a CAR is introduced into cells, e.g., T cells or NK cells, e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of CAR-expressing cells, e.g., CAR T cells or CAR-expressing NK cells of the invention, and one or more subsequent administrations of the CAR-expressing cells, e.g., CAR T cells or CAR-expressing NK cells of the invention, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration.
  • more than one administration of the CAR-expressing cells are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the CAR-expressing cells, e.g., CAR T cells or CAR-expressing NK cells of the invention are administered per week.
  • the subject receives more than one administration of the CAR-expressing cells, e.g., CAR T cells per week or CAR- expressing NK cells (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no CAR-expressing cells, e.g., CAR T cell administrations or CAR-expressing NK cell administrations, and then one or more additional administration of the CAR-expressing cells, e.g., CAR T cells or CAR-expressing NK cells (e.g., more than one administration of the CAR-expressing cells, e.g., CAR T cells or CAR-expressing NK cells, per week) is administered to the subject.
  • the CAR-expressing cells e.g., CAR T cells per week or CAR-expressing NK cells (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle)
  • a week of no CAR-expressing cells e.g., CAR
  • the subject receives more than one cycle of CAR-expressing cells, e.g., CAR T cells or CAR-expressing NK cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days.
  • the CAR-expressing cells e.g., CAR-T cells or CAR-expressing NK cells
  • the CAR-expressing cells e.g., CAR T cells or CAR-expressing NK cells of the invention, are administered for at least two, three, four, five, six, seven, eight or more weeks.
  • CAR-expressing cells as disclosed herein can be administered or delivered to the subject via a biopolymer scaffold, e.g., a biopolymer implant.
  • Biopolymer scaffolds can support or enhance the delivery, expansion, and/or dispersion of the CAR-expressing cells described herein.
  • a biopolymer scaffold comprises a biocompatible (e.g., does not substantially induce an inflammatory or immune response) and/or a biodegradable polymer that can be naturally occurring or synthetic.
  • biopolymers include, but are not limited to, agar, agarose, alginate, alginate/calcium phosphate cement (CPC), beta-galactosidase (P-GAL), (1 ,2,3,4,6-pentaacetyl a-D-galactose), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3 -hydroxy -hexanoate) (PHBHHx), poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO), poly(lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPO), polyvinyl alcohol) (PVA), silk, soy protein, and soy protein isolate, alone or in combination with any other polymer composition, in any concentration and in any other poly
  • the biopolymer can be augmented or modified with adhesion- or migration-promoting molecules, e.g., collagen-mimetic peptides that bind to the collagen receptor of lymphocytes, and/or stimulatory molecules to enhance the delivery, expansion, or function, e.g., anti-cancer activity, of the cells to be delivered.
  • adhesion- or migration-promoting molecules e.g., collagen-mimetic peptides that bind to the collagen receptor of lymphocytes, and/or stimulatory molecules to enhance the delivery, expansion, or function, e.g., anti-cancer activity, of the cells to be delivered.
  • the biopolymer scaffold can be an injectable, e.g., a gel or a semi-solid, or a solid composition.
  • CAR-expressing cells described herein are seeded onto the biopolymer scaffold prior to delivery to the subject.
  • the biopolymer scaffold further comprises one or more additional therapeutic agents described herein (e.g., another CAR-expressing cell, an antibody, or a small molecule) or agents that enhance the activity of a CAR-expressing cell, e.g., incorporated or conjugated to the biopolymers of the scaffold.
  • the biopolymer scaffold is injected, e.g., intratumorally, or surgically implanted at the tumor or within a proximity of the tumor sufficient to mediate an anti-tumor effect. Additional examples of biopolymer compositions and methods for their delivery are described in Stephan et al., Nature Biotechnology, 2015, 33:97
  • An anti-CD72 nanobody of the present disclosure (or antibody or antigen binding molecule comprising the nanobody), or immune effector cells genetically modified to express a nanobody as described herein may be administered with one or more additional therapeutic agents, e.g., radiation therapy, chemotherapeutic agents and/or immunotherapeutic agents.
  • administered "in combination” means that two (or more) different treatments are delivered to the subject for the treatment of the B-cell malignancy, e.g., the two or more treatments are administered after the subject has been diagnosed with the B-cell malignancy.
  • there may be overlap in the time frames in which the two therapeutic agents are administered.
  • one treatment protocol ends before the second begins..
  • treatment may be more effective because of combined administration.
  • the nanobody or immune effector cells that express a CAR comprising the nanobody are administered in conjunction with an agent that targets an immune checkpoint antigen.
  • the agent is a biologic therapeutic or a small molecule.
  • the agent is a monoclonal antibody, a humanized antibody, a human antibody, a fusion protein or a combination thereof.
  • the agents inhibit, e.g., by blocking ligand binding to receptor, a checkpoint antigen that may be PD1, PDL1, CTLA-4, ICOS, PDL2, IDO1, IDO2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, GITR, HAVCR2, LAG3, KIR, LAIR1, LIGHT, MARCO, OX-40, SLAM, , 2B4, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137 (4-1BB), CD160, CD39, VISTA, TIGIT, a SIGLEC, CGEN- 15049, 2B4, CHK 1, CHK2, A2aR, B-7 family ligands or a combination thereof.
  • a checkpoint antigen that may be PD1, PDL1, CTLA-4, ICOS, PDL2, IDO1, IDO2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9,
  • the agent targets PD-1, e.g., an antibody that blocks PD-L1 binding to PD-1 or otherwise inhibits PD-1.
  • agent targets CTLA-4.
  • the agents targets TIM3.
  • the agents target ICOS.
  • the anti-CD72 nanobody or immune effector cells expressing a CAR comprising the nanobody can be administered in conjunction with an additional therapeutic antibody that targets an antigen on a B-cell malignancy.
  • therapeutic antibodies for the treatment of B-cell malignancies include antibodies that target CD20, CD22, and CD19, including, e.g., rituximab, obinutuzumab, tositumomab ofatumumab, veltuzumab, and ocrelizumab. epratuzumab, and blinatomomab.
  • the anti-CD72 nanobody or immune effector cells comprising the antibody are administered with a chemotherapeutic agent.
  • cancer chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, pre
  • paclitaxel and doxetaxel paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; docetaxel, platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as bexarotene, alitretinoin; denileukin diftitox; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above
  • anti-CD72 nanobody or immune effector cells expressing a CAR comprising the nanobody can be administered in conjunction with an additional therapeutic compound that modulates the B-cell receptor signaling complex or other members of its signaling pathway.
  • additional therapeutic compound that modulates the B-cell receptor signaling complex or other members of its signaling pathway.
  • Such compounds include agonists or antagonists of Protein Kinase C, PI3K, BTK, BLNK, PLC-gamma, PTEN, SHIP1, SHP1, SHP2, ERK, and others.
  • therapeutic compounds that target B-cell receptor signaling and/or other members of its signaling pathway include Bryostatin 1, 3AC, RMC-4550, and SHP099.
  • Affinity maturation was performed based on the initial sequence of the nanobody clone NbD4 (WO2022/150389) to improve performance of anti-CD72 nanobodies in CAR-T format.
  • a site- saturation library of NbD4 variants, fused to the Aga2 protein on the surface of yeast was generated ande used to select variants with increased affinity for CD72.
  • overlapping oligos were generated that together recapitulated the entire NbD4 sequence, but incorporated dNTP analogues in every residue position of all three CDR regions while leaving nanobody framework regions unmodified. Assembly of the library using overlap-extension PCR generated a variant library that at most generates one mutation per CDR at a time.
  • Transformation into the EBY100 yeast strain using electroporation resulted in ⁇ 5e8 yeast transformants that were expanded for subsequent yeast display selections.
  • Four equilibrium sorts were performed with increasing stringency, starting at a lOnM concentrations of recombinant CD72 protein and ending at lOOpM, using a combination of MACS and FACS sorting to select higher affinity clones.
  • MACS and FACS sorting In order to isolate the highest affinity clones with a low off-rate, two additional selections were performed using 6-hr and 10-hr off-rate sorts with stringent FACS gating on the top 1% of binders.
  • Yeast clones were isolated and sequenced. Unique sequences resulting from this affinity maturation process were cloned from yeast and characterized, including the sequence NbD4.13 (FIG. 1).
  • Biolayer interferometry was performed to biophysically validate the increased affinity of NbD4.13 sequence compared to the parental NbD4 clone and to the humanized (but not affinity matured) H24 clone.
  • DNA encoding the extracellular domain of CD72 (amino acids 117-359) was PCR-amplified from a plasmid obtained from the Human ORFeome collection (hORFeome 8.1) and cloned into a mammalian expression vector to generate a construct in which the extracellular domains was fused to the C-terminus of a human constant CH2-CH3 domain (Fc domain), along with a N-terminal Avidity AviTag to facilitate site-specific biotinylation during expression.
  • plasmid For expression, 30 pg of plasmid was transiently transfected into Expi293F cells (A14527, modified to stably express ER-localized BirA; Thermo Fisher Scientific). Cells were cultured in Expi293 Expression Medium (A1435101; Thermo Fisher) supplemented with 100 pmol/L biotin for 5 to 7 days to allow for protein expression and biotinylation. Biotinylated CD72 Fc-fusion protein was purified from the cells. Non-biotinylated nanobody-Fc fusions were similarly purified after expression in unmodified Expi293 cells lacking BirA expression.
  • the concentration of Fc- fusion protein was determined by A280 by NanoDrop (Thermo), and molecular weight was confirmed by SDS-PAGE.
  • Bio-layer interferometry data was obtained using an Octet RED384 (ForteBio) instrument. Biotinylated CD72 protein was loaded onto a streptavidin biosensor until 0.2-nm signal was achieved. After blocking with lOuM biotin, each of the nanobody binders was added to determine binding affinity. PBSTB was used as a buffer for all analytes. Data were analyzed using the ForteBio Octet analysis software and kinetic parameters determined with a 1 : 1 monovalent binding model. The assay was performed for NbD4 parental clone, H24 humanized clone, and NbD4.13 affinity matured clone (FIG. 2).
  • FIG. 1 shows flow cytometry data for CD72 expression across several B-cell lymphoma cell lines (Toledo, Namalwa, JeKo-1, Raji; compared to unstained control).
  • FIG. 3B shows in vitro cytotoxicity assay of the resulting CAR-T cells incubated with Toledo or Namalwa B-cell lymphoma cell line.
  • the affinity matured clones were next evaluated to determine if they exhibit improved functionality versus tumor cells carrying low antigen density of CD72.
  • CRISPR- Cas9 technology was used to knock out CD72 from the JeKo-1 lymphoma cell line to generate a model lackingl CD72 surface expression. This line was then further modified to exogenously express CD72.
  • This isogenic model led to a cell line with some CD72 at the cell surface, but ⁇ 3.7x lower expression than WT JeKo-1 based on mean fluorescence intensity (MFI) (FIG. 4A).
  • MFI mean fluorescence intensity
  • NbD4.13-based CAR-T cells showed greater cytokine secretion in response to the JeKo-1 CD72 antigen low cell line and faster tumor clearance in vitro (FIG. 7A-C).
  • FIG. 7 A shows that NbD4.13 CAR-T cells secreted more cytokines than H24-based CAR-T cells when cocultured with low JeKO-1 cells.
  • FIGS. 7B and 7C show the results of a repetitive stimulation assay using the JeKo-1 -low cell line at 1 : 1 effector cell to tumor cell ratio.
  • NbD4.13 CAR-T cells exhibited faster kinetics for killing tumor cells than H24 CAR T cells during 24 hours on second and fifth restimulation with tumor cells.
  • H24 CAR T cells and NbD4.13-H24 -CAR T cells exhibited similar kinetics of killing.
  • the affinity matured, humanized CAR -T cells additionally conferred a survival benefit versus JeKo-1 -CD72 antigen low model (FIGS 8A-D).
  • the group of mice injected with NbD4.13 CAR-T cells showed a significantly lower tumor burden compared to the group injected with H24 CAR-T cells up to 28 days after tumor injection.
  • JeKo-1 low tumor cell line was generated with a first CRISPR/Cas9 knock out of CD72 in parental JeKo-1 cells and subsequent lentiviral reexpression of exogeneous CD72 at MFI 4.7 times less than JeKo-1 parental cell line.
  • CAR-T cells were cocultured with JeKo-1 low cell line at 1 : 1 effector to tumor ratio and then restimulated with tumor cell line every 3 days for a total of 5 times.
  • CAR-T cells were cocultured with tumor after l nd and 4 th stimulation for 60 hours, in triplicates and cytotoxicity was measured using the Incucyte Live-Cell Analysis system (Sartorius). Viable cells were normalized to the initial time point.
  • Control (Empty) and CD72 CAR-Ts were cocultured with parental JeKo-1 or JeKo- 1 low cell lines for 24 hours.
  • Supernatant was diluted with RPMI 20% FBS media at 1 : 1 and snap frozen in liquid nitrogen.
  • Supernatant was evaluated at Eve Technologies by xMAP multiplexed quantification of 14 cytokines.
  • the multiplexing analysis was performed using the LuminexTM 200 system (Luminex, Austin, TX, USA) by Eve Technologies Corp. (Calgary, Alberta). Fourteen markers were measured using Eve Technologies' Human High Sensitivity 14-Plex Discovery Assay® (Millipore Sigma, Burlington, Massachusetts, USA) according to the manufacturer's protocol.
  • the 14-plex consisted of GM-CSF, IFNy, IL-ip, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-23, TNF-a.
  • Assay sensitivities of these markers range from 0.11 - 3.25 pg/mL for the 14-plex.
  • Individual analyte sensitivity values are available in the MilliporeSigma MILLIPLEX® MAP protocol.
  • mice All animal experiments were authorized by the UCSF Institutional Animal Care and Use Committee (IACUC). Male and female NSG ,C -Prkdc scld Il2r ⁇ mlW]l I zi, Jackson Laboratories) strain mice, 8-10 weeks old, were injected via tail-vein with 0.5 million JeKo- 1- low tumor cells stably expressing luciferase, 7 days after control (Empty) CAR or CD72 CAR-Ts were injected, 5 mice per arm. Tumor burden was assessed by weekly bioluminescence imaging. Blood samples were collected 9 days after CAR injection to assess CAR-T peripheral expansion by flow cytometry staining with human CD3 antibody. Survival endpoint was when mice demonstrated signs of illness.
  • IACUC Institutional Animal Care and Use Committee
  • Anti-CD72 nanobody polypeptide sequences are:

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Abstract

L'invention concerne des nanocorps anti-CD72 et des procédés d'utilisation de tels nanocorps à des fins diagnostiques et thérapeutiques.
PCT/US2024/054950 2023-11-10 2024-11-07 Nanocorps anti-cd72 améliorés pour immunothérapie Pending WO2025216764A2 (fr)

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