Improved Anti-cd72 nanobodies for immunotherapy

Abstract

Provided herein are anti-CD72 nanobodies and methods of using such nanobodies for diagnostic and therapeutic purposes.

Description

Improved Anti-CD72 Nanobodies for ImmunotherapyCROSS-REFERENCE TO RELATED APPICATIONS

[0001] This claims priority benefit to U.S. Provisional Application No. 63 / 597,819, filed November 10, 2023, which is incorporated by reference for all purpose] s.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under grant no. W81XWH2210807 awarded by the Department of Defense. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] 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.BRIEF SUMMARY OF ASPECTS OF THE DISCLOSURE

[0004] 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). In some embodiments, 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 sequenceTYYADSVKGRFTISRDNSQNTLYLQMNSLRAEDTAVYYCA (SEQ ID N0:7), and an FR4 sequence YWGQGTQVTVSS (SEQ ID N0:8).

[0005] In some embodiments, 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. In some embodiments, the FR3 comprises SEQ ID NO:7. In some embodiments, 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. In some embodiments, the nanobody comprises SEQ ID NO: 1.

[0006] In a further aspect, provided herein is 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, the method 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. In some embodiments, the hematological malignancy is a B-cell leukemia, e.g., chronic lynmphocytic leukemia. In some embodiments, the hematological malignancy mixed-lineage leukemia (MLL). In some embodiments, 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.

[0007] In a further aspect, provided herein is a method of treating an autoimmune disease, the method 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.

[0008] In addition, the disclosure provides a polynucleotide encoding an anti-CD72 nanobody as described here. In further embodiments, the disclosure provides a polynucleotide encoding a CAR comprising one or more anti-CD72 nanobody of the present invention. Further, the disclosure provides vectors comprising such polynucleotides andmammalian host cells, e.g., immune effector cells, comprising the polynucleotides. In some embodiments, the vector a retroviral vector, e.g., a self-inactivating lentiviral vector. In some embodiments, the immune effector cell is a T lymphocyte or NK cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] 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.

[0010] FIG. 2. Bio-layer interferometry graphs showing binding of NbD4, H24 and NbD4.13 nanobody to biotinylated CD72 protein. Data from two technical replicates. Best-fit kon, koir, and KD are shown. NbD4.13 exhibited improved binding. Best-fit kon, koff, and KD are shown. NbD4.13 exhibited improved binding (KD = 0.78 nM for NbD4.13 vs. 50.4 nM for NbD4 and 33.0 nM for H24).

[0011] FIG. 3A-B. CAR-T cells generated with affinity -matured CD72 nanobodies demonstrate superior anti-tumor potency to H24. A) CD72 antigen expression measured in lymphoma cell lines by flow cytometry. B) In vitro cytotoxicity assays comparing H24, NbD4.13, and humanized NbD4.13 CAR-T cells against Toledo (24-hour exposure) and Namalwa (48-hour exposure) at 1 : 1, 1 :3 and 1 : 10 effector to tumor ratios (n=4 technical replicates). P value was determined by a two-tailed unpaired Student's t-test, **=p< 0.01

[0012] FIG. 4A-B. CAR-T cells generated with affinity -matured CD72 nanobodies demonstrate superior killing of JeKo-CD72 antigen low cell line. A) CD72 “low” antigen model was produced by transducing clonal CD72 KO JeKo-1 cell line with recombinant CD72 construct, with MFI of CD72 expression decreasing 4.7 times compared to wild type cell line. B) 24-h in vitro cytotoxicity assay of affinity matured clones NbD4.13 and humanized NbD4.13 CAR vs JeKol-CD72 “low” model (n=4 technical replicates). P values were determined by a two-tailed unpaired Student's t-test, **** = p < 0.0001

[0013] FIG. 5A-D Affinity matured NbD4.13 significantly prolongs survival in vivo compared to H24 in mantle cell lymphoma model. A) NSG Mice were injected with tumor on day -7 and then with CAR-Ts on day 0, 5 mice per group. B) Tumor burden was monitored by weekly BLI imaging of mice, average radiance, photons / second is shown. C) Kaplan- Meier survival curves of mice treated with empty CAR, H24 and NbD4.13 CAR. P valuesobtained with the log-rank (Mantel-Cox) test * = p < 0.05. D) Quantification of blood CAR- T expansion in mouse peripheral blood, measured by flow cytometry.

[0014] 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. A) JeKo-1 CD72 antigen “low” tumor was isolated from spleens of animals relapsed after H24 CAR-T treatment. CD72 molecules per cell was estimated on JeKo-1 WT and Empty CAR and H24 treated relapsed tumor cells with quantification beads by flow cytometry. B) 24-h incucyte assay of H24 and NbD4.13 CAR vs. post-relapse Jekol tumor with reduced CD72 expression at 1 : 10 effector to tumor ratio, mcherry positive tumor total red object intensity normalized to initial time point. P values were determined by a two-tailed unpaired Student's t-test, ***p < 0.001, ** = p < 0.01

[0015] 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. A) Cytokine profiling versus JeKo-1 wild type and JeKo-1 -low cell lines. B), C) Repetitive stimulation assay with JeKo-1 -low cell line at 1 : 1 effector to tumor ratio.

[0016] FIG. 8A-D Affinity matured, humanized CAR conferred a survival benefit versus JeKo-1 -CD72 antigen low model. A) NSG mice were implanted with JeKo-1 -CD72 low tumor cells at 0.5 e6 cells per mouse and 1 week later with 5e6 CAR-T cells as indicated for 5 groups. Tumor burden measurements in average radiance, photons / second in mice were evaluated weekly with BLI. n=5 / arm B) Flow plots of blood CAR-T measurements on day 9 after CAR injection C) CAR-T positive cell percentage plot, corresponding to day 9 after CAR injection D) Survival curve of mice treated with Empty CAR, H24 CAR, NbD4.13 CAR and NbD4.13-H24 CAR. P values obtained with the log-rank (Mantel-Cox) test *p=0.0442.DETAILED DESCRIPTION OF THE DISCLOSURETerminology

[0017] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells andreference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

[0018] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. For example, for KD and IC50 values ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0019] “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.

[0020] The term "nanobody" as used herein 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). As further detailed below, in some embodiments, 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). The term “nanobody” includes humanized versions of nanobodies.

[0021] As used herein, “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.

[0022] As used herein, "complementarity-determining region (CDR)" refers to the three hypervariable regions (HVRs) that interrupt the four "framework" regions of s variable domain. The CDRs are the primary contributors to binding to an epitope of an antigen. The CDRs of are referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. The term “CDR” may be used interchangeably with “HVR”.

[0023] 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)).Definitions of 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. USA, 86, 9268-9272 (1989); Martin, et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996). Reference to CDRs as determined by Kabat numbering are based, for example, on Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are determined as defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0024] “Epitope" or "antigenic determinant" refers to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguousamino 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).

[0025] The term "valency" as used herein 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.

[0026] The phrase “specifically (or selectively) binds” to an antigen or target or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction whereby the antibody binds to the antigen or target of interest. In the context of this invention, the antibody binds to CD72 with a KD that is at least 100-fold greater than its affinity for other antigens.

[0027] The terms “identical” or percent “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. Examples examples of algorithms that are suitable for determining percent sequence identity and sequence similarity the BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.215:403-410 (1990). Thus, for purposes of this invention, BLAST 2.0 can be used with the default parameters to determine percent sequence identity.

[0028] The terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence, refers to the position of the residue of a specified reference sequencewhen the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, 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.

[0029] 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.

[0030] The terms “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. In particular embodiments, 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. In addition, a polynucleotide, e.g., a cDNA or mRNA, 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.). The above term is also intended to include any topological conformation, including single-stranded, double-stranded, partially duplexed,triplex, hairpinned, circular and padlocked conformations. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, 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.

[0031] The term "vector," as used herein, 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".

[0032] The terms “subject”, “patient” or “individual” are used herein interchangeably to refer to any mammal, including, but not limited to, a human. For example, 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. In some embodiments, the subject”, “patient” or “individual” is a human.Anti-CD72 Nanobodies

[0033] Provided herein are anti-CD72 binding domains that can be used for diagnostic and therapeutic purposes.

[0034] In some embodiments, 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.

[0035] In some embodiments, 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.

[0036] In some embodiments, 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. In some embodiments, the variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in SEQ ID NO: 1. In some embodiments, 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. In this context, 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.

[0037] In some embodiments, 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.

[0038] In some embodiments, 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.

[0039] In some embodiments, 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.

[0040] In some embodiments, 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.

[0041] In some embodiments, 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. In some embodiments, 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 andR 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. In some embodiments, the nanobody comprises SEQ ID NO: 1.Antibody formats

[0042] As previously explained, 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. In some embodiments, 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. In some embodiments, such an antibody may comprise an Fc region. In some embodiments, 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. In further embodiments, 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.

[0043] In some embodiments, 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. Thus, for example, in some aspects, 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. Accordingly, in some embodiments, 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. In some embodiments, a bispecific antibody comprises an anti-CD72 nanobody as described herein and an antibody, e.g., scFV, that targets CD28.

[0044] In some embodiments any of the nanobodies or antibodies described herein are linked to a toxin or drug, e.g., to form an antibody-drug conjugate (ADC) . In certain embodiments, the nanobody is attached to a therapeutic cytotoxic / cytostatic drug. In various embodiments 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. In certain embodiments 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).CAR constructs comprising an anti-CD72 nanobody

[0045] Chimeric antigen receptors (CARs) 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. In certain embodiments, immune cells (e.g., T cells or natural killer (NK) cells) are genetically modified to express CARs that comprise one or more anti-CD72 nanobodies of the present and have the functionality of effector cells (e.g., cytotoxic and / or memory functions of T cells or NK cells).

[0046] In a standard CAR, 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. In the present invention, 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). For example, 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.Extracellular domain (Nanobody domain)

[0047] 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. In some embodiments, 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.

[0048] In some embodiments, the extracellular domain may comprise two more anti-CD72 nanobodies. For example, the extracellular domain may comprise three of four different nanobodies that are described herein. In some embodiments, the extracellular domain may comprises multiple copies of the same nanobody. In some embodiments, the extracellular domain may comprise a nanobody as described herein and an anti-CD72 nanobody that binds to a different CD72 epitope. In some embodiments at least one of the nanobodies comprises a CDR1 sequence comprising TISPIDQ, a CDR2 sequence comprising FVAAIALGGN or and a CDR3 sequence comprising VGYVDKWDDSNYHT.

[0049] 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.Hinge domain

[0050] In some embodiments, 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. Such 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 domain

[0051] Any 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. In some embodiments, 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, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100, (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD 150, IPO-3), BLAME, (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C.

[0052] A transmembrane domain incorporated into a CAR construct may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.Intracellullar signaling domain

[0053] 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. In one embodiment, a co-stimulatory domain is used that increases CAR immune T cell cytokine production. In another embodiment, a co- stimulatory domain is used that facilitates immune cell (e.g., T cell) replication. In still another embodiment, a co-stimulatory domain is used that prevents CAR immune cell (e.g., T cell) exhaustion. In another embodiment, a co-stimulatory domain is used that increases immune cell (e.g., T cell) antitumor activity. In still a further embodiment, a co-stimulatory domain is used that enhances survival of CAR immune cells (e.g., T cells) (e.g., post-infusion into patients).

[0054] Examples of 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.

[0055] 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.

[0056] 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), CD3gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In one embodiment, a CAR comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3- zeta.

[0057] 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. For example, 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. Examples of such molecules include CD27, CD28, 4- IBB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds to CD83, and the like. For example, 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). Further examples of such 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, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM, (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD 19a.

[0058] In some embodiments, 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. In some embodiments, the CAR may comprise a safety switch gene. In some emboidments, 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).

[0059] In some embodiments, the CAR-expressing cell uses a split CAR. The split CAR approach is described in more detail in publications WO2014 / 055442 and WO2014 / 055657. Briefly, 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). When the cell encounters the first antigen, the costimulatory domain is activated, and the cell proliferates. When the cell encounters the second antigen, the intracellular signaling domain is activated and cell-killing activity begins. Thus, the CAR- expressing cell is only fully activated in the presence of both antigens.

[0060] In some embodiments, a host cell, e.g., a T cell, can be engineered such that a synthetic Notch receptor comprising an extracellular domain that targets one antigen induces the expression of a CAR that targets a second antigen. Such systems are described, e.g., in U.S.. Patent Application Publication No. 20190134093; see also, synNotch polypeptides as described in US20160264665, each incorporated herein by reference. In some embodiments, a synNotch comprises a one or more anti-CD72 nanobodies as described herein. In some embodiments, 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.

[0061] In some embodiments, 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.

[0062] In some embodiments, 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. Science Translational Medicine 13(586):eabb5191, 2021), antibody-T-cell receptor (AbTCR) (e.g., those disclosed in Xu et al. Cell Discovery (2018) 4:62), and T cell antigen coupler (TAC) (e.g., those disclosed in Helsen et al. Nature Communications (2018);9:3049).Activation and Expansion of Immune Effector Cells (e.g., T Cells)

[0063] 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. In some embodiments, the immune cells, e.g., T cells, are autologous cells from the patient to undergo immunotherapy. In some embodiments, the immune cells are allogeneic.

[0064] 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. Examples of 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.

[0065] Methods of making CAR-expressing cells are described, e.g., in US2016 / 0185861 and US2019 / 0000880.Nucleic Acids and Vectors Encoding CARS

[0066] 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. Nonlimiting examples of 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.22:223-230 (1994); and Hughes, et al. J. Clin. Invest. 89: 1817 (1992).

[0067] In some embodiments, genetic modification is performed using transposase-based systems for gene integration, CRISPR / Cas-mediated gene integration, TALENS or Zinc- finger nucleases integration techniques. For example, 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.Nanobody Conjugates

[0068] In a further aspect, an anti-CD72 nanbody of the present disclosure may be conjugated or linked, either directly or indirectly, to therapeutic and / or imaging / detectable moieties. For example, in some embodiments, 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. Methods for conjugating or linking a nanobody, or antigen binding regions comprising a nanobody, to a desired molecule moiety are well known in the art. The moiety may be linked to the nanobody covalently or by non-covalent linkages.

[0069] In some embodiments, an anti-CD72 nanobody of the present invention, or an antigen binding domain comprising an anti-CD72 nanobody of the present invention, is conjugated to cytotoxic moiety or other moiety that inhibits cell proliferation. In some embodiments, 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, restrictocin, cobran venom factor, a ribonuclease, engineered Shiga toxin, phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolastatin, ccl065, or a cisplatin. In some embodiments, 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.

[0070] In some embodiments, 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. In some embodiments, 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.Cancer Vaccines

[0071] 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). 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.

[0072] In some embodiments, 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. Examples of 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. In some embodiments, the complex may be coadministered with an adjuvant, such as a Toll-like receptor (TLR) agonist, a C-type lectinreceptor (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.Treatment of B-cell malignancies

[0073] 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. In some embodiments, the malignancy is a B cell malignancy. Illustrative 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, extranodal marginal zone lymphoma of mucosa- associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, follicular lymphoma, In situ follicular neoplasia, duodenal- type follicular lymphoma, pediatric-type follicular lymphoma, large B-cell lymphoma with IRF4 rearrangement, primary cutaneous follicle center lymphoma, mantle cell lymphoma, in situ mantle cell neoplasia, diffuse large B-cell lymphoma (DLBCL) NOS, including germinal center B-cell type, and activated B-cell type; T-cell / histiocyte-rich large B-cell lymphoma, primary DLBCL of the central nervous system, primary cutaneous DLBCL, leg type, EBV+DLBCL NOS, EBV+mucocutaneous ulcer, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, HHV8+DLBCL NOS, Burkitt lymphoma, Burkitt- like lymphoma with 1 Iq aberration, high-grade B-cell lymphoma, with MYC and BCL2 and / or BCL6 rearrangements, high-grade B-cell lymphoma NOS, and B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin lymphoma. In some embodiments, a malignancy treated with an anti-CD72 nanobody as described hereinis 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. In some embodiments a malignancy treated with an anti-CD72 nanobody as described herein in a posttransplant lymphoproliferative disorder (PTLD), 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. In some embodiments, 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.

[0074] In some embodiments, 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. 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.Treatment of autoimmune disease

[0075] 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. Illustrative 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, polymyositis, interstitial lung disease, Crohn’s disease, ulcerative colitis, thyroid autoimmune disease, autoimmune uveitis, undifferentiated connective tissue disease, discoid lupus erythematosus, an immune-mediated inflammatory disease (IMID) such as scleroderma, rheumatoid arthritis or Sjogren’s disease, an autoimmune connective tissuedisease such as systemic lupus erythematosus, graft versus host disease, mixed connective tissue disease, atopic asthma, atopic dermatitis, Churg-Strauss vasculitis, allergic rhinitis, allergic eye disease, chronic non-autoimmune urticaria, and eosinophilic oesophagitis.Administration of anti-CD72 nanobody

[0076] In one aspect, 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.

[0077] 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. In some embodiments 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.

[0078] 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. In any event, the methods provide asufficient quantity of anti-CD72 nanobody or antigen binding molecule that comprises the anti-CD72 nanobody to effectively treat the patient.

[0079] 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. In some embodiments, the nanobody may be administered by insufflation. In an illustrative embodiment, 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. In some embodiments, the nanobody is administered by intravenous infusion over the course of 1 hour at a dose of between 0.01 and 25 mg / kg. In other embodiments, the nanobody is administered by intravenous infusion over a period of between 15 minutes and 2 hours. In still other embodiments, the administration procedure is via sub-cutaneous bolus injection.

[0080] 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. Preferably 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). In some embodiments, 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. In other embodiments, the nanobody is administered approximately once per month.Administration of Immune Effector Cells comprising an Anti-CD72 nanobody

[0081] In some embodiments, pharmaceutical 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 suchas glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, immune effector cells genetically modified to express a CAR comprising an anti-CD72 nanobody are formulated for intravenous administration.

[0082] Pharmaceutical 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.

[0083] In some embodiments, 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 104to 109cells / kg body weight, in some instances 105to 106cells / kg body weight, including all integer values within those ranges. In some embodiments, the cells, e.g., T cells or NK cells modified as described herein, may be administered at 3 x 104, l x 106, 3 x 106, or 1 x 107cells / kg body weight. The cell compositions may also be administered multiple times at these dosages. Administration can be performed using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). In some embodiments, the genetically modified immune effector cells are administered intravenously. In 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.

[0084] In some embodiments, 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. 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. In certain aspects, following or concurrent with the transplant, subjects receive an infusion of the expanded CAR-expressing cells of the present invention. In an additional aspect, expanded cells are administered before or following surgery.

[0085] In embodiments, 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.

[0086] In one embodiment, 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. In one embodiment, more than one administration of the CAR-expressing cells, e.g., CAR T cells or CAR-expressing NK cells of the invention 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. In one embodiment, the subject (e.g., human 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. In another embodiment, the subject (e.g., human 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. In one embodiment, the CAR-expressing cells, e.g., CAR-T cells or CAR-expressing NK cells, are administered every other day for 3 administrations per week. In one embodiment, 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.

[0087] In some embodiments, 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. Examples of suitable 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 ratio. 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. The biopolymer scaffold can be an injectable, e.g., a gel or a semi-solid, or a solid composition.

[0088] In some embodiments, CAR-expressing cells described herein are seeded onto the biopolymer scaffold prior to delivery to the subject. In embodiments, 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. In embodiments, 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, 33:97Administration in combination with other agents

[0089] 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. As used herein, 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. In some embodiments, there may be overlap in the time frames in which the two therapeutic agents are administered. In other embodiments, one treatment protocol ends before thesecond begins.. In some embodiment, treatment may be more effective because of combined administration.

[0090] In some embodiments, 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. In one aspect, the agent is a biologic therapeutic or a small molecule. In another aspect, the agent is a monoclonal antibody, a humanized antibody, a human antibody, a fusion protein or a combination thereof. In certain embodiments, 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. In some embodiments, the agent targets PD-1, e.g., an antibody that blocks PD-L1 binding to PD-1 or otherwise inhibits PD-1. In some embodiments, agent targets CTLA-4. In some embodiments, the targets LAG3. In some embodiments, the agents targets TIM3. In some embodiments, the agents target ICOS.

[0091] In some embodiments, 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. Examples of 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.

[0092] In some embodiments, the anti-CD72 nanobody or immune effector cells comprising the antibody are administered with a chemotherapeutic agent. Examples of 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, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norieucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5- fluorouracil; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoids, e.g. 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. In some embodiments, 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. Such compounds include agonists or antagonists of Protein Kinase C, PI3K, BTK, BLNK, PLC-gamma, PTEN, SHIP1, SHP1, SHP2, ERK, and others. Examples of therapeutic compounds that target B-cell receptor signaling and / or other members of its signaling pathway include Bryostatin 1, 3AC, RMC-4550, and SHP099.

[0093] The following examples illustrate certain aspects of the claimed invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.Technical sectionBackground

[0094] 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. Specifically, 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. 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).

[0095] Humanization of the NbD4.13 sequence was performed as previously described (see, WO2022150389), specifically modulating framework residues of the nanobody to specific human residues. Humanized derivatives were generated based on the human variable-heavy chain framework sequence of the clinically approved anti-Her2 antibody Trastuzumab (Herceptin). The thirteen residue positions that varied between llama and human VH framework were systematically exchanged with their human counterparts indifferent permutations. Llama residues Y37, E44, and R45 in framework region 2 were retained as this region, as they have been shown to play a role in the monomeric nature of nanobodies. The resulting sequence, incorporating both 9 humanized framework residues as well as specific CDR mutations from affinity maturation (“humanized NbD4.13”) is provided in FIG. 1.

[0096] 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. 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).

[0097] The noted anti-CD72 nanobody sequences (FIG. 1) were introduced into a CAR-T backbone (including mutated IgG4 hinge domain (“EQ”), CD28 transmembrane sequence, CD28 co-stimulatory domain, and CD3 zeta domain) and evaluated for in vitro cytotoxicity versus CD72-expressing B-cell malignancy cell lines. FIG. 3 A 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 theresulting CAR-T cells incubated with Toledo or Namalwa B-cell lymphoma cell line. These results show that in vitro, after either 24 hour (Toledo) or 48 hour (Namalwa) incubation, across a range of Effector: Turn or (i.e. CAR-T:tumor cell line) ratios, the “humanized NbD4.13” affinity matured binder consistently provided higher levels of lysis than the “NbD4” clone or “NbD4.13” affinity matured from without humanization. Notably, all clones lead to significantly higher lysis than the “empty” CAR-T (i.e. no antigen recognition element) control.

[0098] 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). In 24 hour cytotoxicity assays at a range of CAR-T doses (FIG. 4B), against this CD72 “low” JeKo-1 cell line, it was found that the humanized NbD4.13-based CAR-T had superior tumor lysis activity when compared to the unhumanized NbD14.13 sequence. Notably, the H24 (humanized but not affinity matured) showed minimal efficacy against in lysis in this CD72 “low” model. These findings are particularly relevant for treatment of patients that have tumors that express lower levels of CD72.

[0099] An in vivo analysis using a murine model that is standard in the field (FIG. 5) was then performed. JeKo-1 tumor cells were administered in an amount of le6 cells intravenously to NOD scid gamma (NSG) immunocompromised mice and then intravenously treated with 3.5e6 CAR-T cells 7 days later. Tumor burden was monitored by biolayer interferometry. Time to murine symptoms necessitating animal sacrifice, per veterinary regulations, was used as a proxy for survival. Improved tumor control and improved survival were observed after treatment with NbD4.13 affinity matured CAR-Ts compared to H24 humanized CAR-Ts. Peripheral blood CAR-T expansion was measured by flow cytometry with staining by human CD3 antibody.

[0100] A separate model of CD72 “low” JeKo-1 tumor was alo generated. Spleens were harvested from NSG mice following relapse on H24-based CAR-T treatment. Flow cytometry demontrated that these recovered tumor cells showed decreased levels of surface CD72, suggesting that antigen downregulation may be a means of resistance to H24-basedCD72 nanoCAR-Ts (FIG. 6A). In vitro culture of these CD72 “low” cells with H24-based CD72 nanoCARs showed minimal tumor lysis; however, NbD4.13-based CAR-Ts were able to more effectively control tumor growth after 24 hours (FIG. 6B). This in vitro assay was performed using an Incucyte live cell imager. Viable cells at 24 hr were normalized to the initial time point.In vivo analyses using JeKo-1 CD72 low cell lines

[0101] 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.

[0102] 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.Methods employing JeKo-1 CD72 antigen low cell line in vivoJeKo-1 CD72 antigen low cell line Incucyte assay

[0103] The JeKo-1 CD72 antigen low (“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 lndand 4thstimulation 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.Cytokine profiling

[0104] 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 andsnap frozen in liquid nitrogen. Supernatant was evaluated at Eve Technologies by xMAP multiplexed quantification of 14 cytokines. The multiplexing analysis was performed using the Luminex™ 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.JeKo-1 -CD72 low cell line mouse study

[0105] All animal experiments were authorized by the UCSF Institutional Animal Care and Use Committee (IACUC). Male and female NSG,C -PrkdcscldIl2r^mlW]lI 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.Statistical analysis

[0106] Statistical analysis was performed in GraphPad Prism version 10.1.1 (270).

[0107] All publications, patent applications, and accession numbers 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 for the material for which it is cited.Anti-CD72 nanobody polypeptide sequences:SEQ ID NO:1 Humanized D4.13 CDR sequences are underlinedEVQLVESGGGLVQPGGSLRLSCAASGTISPIDQMSWYRQAPGKEREFVAAIFLGGNT YYADSVKGRFTISRDNSONTLYLQMNSLRAEDTAVYYCAVGYVDKWDDSNYHTY WGQGTQVTVSSSEQ ID NO:2 CDR1 D4.13 and Humanized D4.13TISPIDQSEQ ID NO:3 CDR2 D4.13 and Humanized D4.113FVAAIFLGGNSEQ ID NO:4 CDR3 D4.13 and Humanized D4.113VGYVDKWDDSNYHTSEQ ID NO:5 FR1 Humanized D4.13EVQLVESGGGLVQPGGSLRLSCAASGSEQ ID NO:6 FR2 Humanized D4.13MSWYRQAPGKERESEQ ID NO:7 FR3 Humanized D4.13TYYADSVKGRFTISRDNSQNTLYLQMNSLRAEDTAVYYCASEQ ID NO:8 FR4 Humanized D4.13YWGQGTQVTVSSSEQ ID NO:9 FR1 D4.13QVQLVESGGGLVQPGGSLRLSCAASGSEQ ID NO: 10 FR2 D4.13MGWYRQAPGKEREFSEQ ID NO:!! FR3 D4.13TYYADSVKGRFTISRDNAQNTVYLQMNSLKAEDTAVYYCASEQ ID NO:8 FR4 D4.13YWGQGTQVTVSSSEQ ID NO: 12 NbD4.13 CDR sequences are underlinedOVOLVESGGGLVOPGGSLRLSCAASGTISPIDQMGWYRQAPGKEREFVAAIFLGGNTYYADSVKGRFTTSRDNAONTVYLQMNSLKAEDTAVYYCAVGYVDKWDDSNYHTY WGQGTQVTVSS

Claims

WHAT IS CLAIMED IS:

1. A nanobody that specifically binds to CD72, wherein the nanobody comprises: a CDR1 sequence comprising TISPIDQ, a CDR2 sequence comprising FVAAIFLGGN, and a CDR3 sequence comprising VGYVDKWDDSNYHT.

2. The nanobody of claim 1, wherein the nanobody binding domain comprises an FR1, an FR2, an FR3, and an FR4 as follows: the FR1 comprises SEQ ID NO:9, or a variant thereof having one amino acid substitution where the variant comprises E at position 1; the FR2 comprises SEQ ID NO: 10, or variant thereof having one amino acid substitution where the variant comprises S at position 35; the FR3 comprises SEQ ID NO: 11 or a variant thereof having 1, 2, or 3 amino aicd substitions where the substitutions are 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.

3. The nanobody of claim 2, wherein the FR1 comprises SEQ ID NO:5; the FR2 comprises SEQ ID NO:6; the FR3 comprises 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.

4. The nanobody of claim 1, wherein the nanobody comprises SEQ ID NO: 1.

5. A bispecific or multispecific antibody comprising a nanobody of any one of claims 1-4.

6. A chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and / or a primary signaling domain, wherein the antigen binding domain comprises a nanobody of any one of claims 1-4.

7. The CAR of claim 6, wherein the antigen binding domain comprises a nanobody comprising SEQ ID NO: 1.

8. The CAR of claim 6 or 7, wherein the CAR is a standard CAR, a split CAR, an off-switch CAR, an on-switch CAR, a first-generation CAR, a second-generation CAR, a third-generation CAR, or a fourth-generation CAR.

9. An immune effector cell comprising a CAR of any one of claims 6 to 8.

10. The immune effector cell of claim 9, wherein the cell is a T lymphocyte or a natural killer (NK) cell.

11. 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, the method comprising administering a plurality of immune effector cells of claim 9 or 10 to a subject that has the hematological malignancy.

12. The method of claim 11, wherein the plurality of immune effector cells comprises allogeneic cells.

13. The method of claim 11, wherein the plurality of immune effector cells comprises autologous cells.

14. The method of claim 11, 12, or 13, wherein the hematological malignancy is a B-cell leukemia.

15. The method of claim 14, wherein the B-cell leukemia is chronic lynmphocytic leukemia.

16. The method of claim 14, wherein the B-cell leukemia is mixed-lineage leukemia (MLL).

17. The method of claim 11, wherein the hematological malignancy is a non-Hodgkin’s lymphoma.

18. The method of claim 11, wherein the hematological malignancy is multiple myeloma.

19. The method of any one of claims 12-18, wherein the hematological malignancy comprises tumor cells having low levels of CD72.

20. The method of any one of claims 11 to 19, wherein the subject is a human.

21. A method of treating an autoimmune disease, the method comprising administering a plurality of immune effector cells of claim 9 or 10 to a subject that has the autoimmune disease.

22. A polynucleotide encoding a CAR of any one of claims 6 to 8.

23. A vector comprising the polynucleotide of claim 22.

24. The vector of claim 23, wherein the vector is a retroviral vector.

25. The vector of claim 24, wherein the retroviral vector is a selfinactivating lentiviral vector.

26. An immune effector cell comprising a vector of any one of claims 23, 24, or 25.

27. The immune effector cell of claim 26, wherein the cell is a T lymphocyte or NK cell.

28. A host cell comprising the polynucleotide of claim 22.

29. The host cell of claim 28, wherein the host cell is an immune effector cell.

30. The host cell of claim 29, wherein the immune effector cell is a T lymphocyte or NK cell.

31. A nucleic acid encoding a nanobody ofof any one of claims 1-4.

32. An expression vector comprising the nucleic acid of claim 31.

33. A host cell comprising the nucleic acid of claim 31.

34. A host cell comprising the expression vector of claim 32.