Patent Application
20240299449
Despite the enormous strides made in the treatment of hematological malignancies, many patients continue to experience poor clinical outcomes. In 2020 alone, its estimated that nearly 178,000 people will be diagnosed with a blood cancer in the United States while >56,000 people will die. (Cancer Facts & Figures, 2020. American Cancer Society; 2020). Although development of new immunotherapies such as CD19 or CD22-directed CAR-T have achieved remarkable initial response rates, a large fraction of these patients eventually relapse. Therefore, new strategies are needed for the treatment of refractory B-cell malignancies, and in particular alternative surface targets to direct CAR-T cells.
Using cell-surface proteomics of B-ALL cell lines and analysis of patient samples to identify cell-surface markers specifically enriched on B-ALL, CD72 was identified as a potential alternative immunotherapy target, orthogonal and complimentary to current CD19 and CD22 directed therapies. CD72 is a highly abundant cell surface protein found enriched on leukemia and lymphoma cells, similar to the canonical B-cell markers CD19 and CD22 that are currently being targeted with immunotherapies in the clinic. Accordingly, provided herein are anti-CD72 nanobodies that can be used for diagnostic and therapeutic purposes, e.g., for the development of CAR-T therapies that target CD72-expressing malignancies.
In one aspect, provided herein is a nanobody that specifically binds to CD72, wherein the nanobody comprises:
In some embodiments, the nanobody comprises:
In some embodiments, the nanobody comprises the CDR1 sequence comprising TISPIDI, the CDR2 sequence comprising (F/L)VAAIALGGN, and the CDR3 sequence comprising VGYVDKWDDSNYHT. In some embodiments, the framework has at least 80% identity to a human antibody heavy chain framework, e.g., a VH3 family member. In some embodiments, the nanobody comprises a framework having at least 80%, or at least 85%, at least 90%, or at least 95%, identity to a framework of comprising an FR1 sequence QVQLQESGGGLVQAGGSLRLSCAASG, an FR2 sequence MGWYRQAPGKERE, an FR3 sequence TYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCA, and an FR4 sequence YWGQGTQVTVSS.
Additionally provided herein is a nanobody that specifically binds to CD72, wherein the nanobody comprises:
Additionally provided herein is a nanobody that specifically binds to CD72, wherein the nanobody comprises:
In some embodiments, the antibody comprises a variable region comprising:
LVAGITSGTN, and a CDR3 sequence comprising AHWPYNQTYT;
FVATITDGAS, and a CDR3 sequence comprising VADPHDYYHH; or
In some embodiments, a nanobody provided herein, e.g., as set forth in the preceding paragraphs of this section, comprises a substitution as determined with reference to SEQ ID NO:6 at one or more positions selected from positions 1, 5, 14, 35, 37, 44, 45, 47, 76, 80, 88, and 89. In some embodiments, the nanobody comprises one, two, three, or four amino acid residues selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89. In some embodiments, the nanobody comprises five, six, seven, eight, nine or ten residues selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89. In some embodiments, the nanobody comprises eleven residues selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89. In some embodiments, the nanobody comprises Q at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, Q at position 44, R at position 45, L at position 47, A at position 76, V at position 80, R at position 88, and A at position 89. In some embodiments, the nanobody comprises Q at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, Q at position 44, R at position 45, F at position 47, A at position 76, V at position 80, K at position 88, and P at position 89. In some embodiments, the nanobody comprises the sequence of antibody D4-H3 or D4-H10 as shown in
In a further aspect, provided herein is 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 an anti-CD72 nanobody as described herein, e.g., in the preceding paragraphs in this section. In some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a costimulatory domain and/or a primary signaling domain, wherein the antigen binding domain comprises a nanobody comprising:
In some embodiments, the antigen binding domain comprises two, three or four nanobodies selected from the group consisting of:
In some embodiments, the antigen binding domain comprises one, two or three nanobodies selected from the group consisting of
In some embodiments, 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.
In a further aspect, provided herein is a synthetic Notch receptor comprising at least one anti-CD72 nanobody that comprises:
VGYVDKWDDSNYHT; or
In some embodiments, the antigen binding domain of the synthetic Notch receptor comprises one, two or three nanobodies selected from the group consisting of
In a further aspect, provided herein is an immune effector cell comprising a CAR or synthetic Notch receptor comprising one or more anti-CD72 nanobodies as described herein, e.g., as described in the preceding paragraphs. In some embodiments, the immune effector cell is a T lymphocyte or a natural killer (NK) cell. In some embodiments, the immune effector cell is an autologous cell from a subject to be treated with the immune effector cell. In other embodiments, the immune effector cell is an allogeneic cell.
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 lymphocytic 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.
Additionally provided herein is 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 and mammalian 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.
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 and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.
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.
“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 9p13.3 (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.
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 the nanobodies as described herein.
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.
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”.
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. January 1;29(1):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)).
“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 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).
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.
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.
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 70%, at least 75%, 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 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.
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 sequence when 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.
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 Val, Leu and Ile; (vi) slightly polar amino acids Met and Cys; (vii) small-side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro; (viii) aliphatic amino acids Val, Leu, Ile, 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.
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 double-stranded 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, internucleotide 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.
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 self-replicating 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. 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.
Provided herein are anti-CD72 nanobodies that can be used for diagnostic and therapeutic purposes.
In some embodiments, an anti-CD72 nanobody of the present disclosure has a KD less than about 10 nM.
In some embodiments, an anti-CD72 nanobody of the invention has at least one, at least two, or three CDRs of a variable domain sequence of any one of SEQ ID NOS: 1-8. In some embodiments, an anti-CD72 nanobody of the present invention comprises a CDR3 selected from the CDR3 sequences of a variable domain sequence of any one of SEQ ID NOS:1-8. In some embodiments, an anti-CD72 nanobody of the present invention comprises a CDR3 selected from the CDR3 sequences of a variable domain sequence of any one of SEQ ID NOS:4, 5, or 6. In some embodiments, an anti-CD72 nanobody of the present invention comprises a CDR3 of a variable domain sequence of SEQ ID NO:6. In some embodiments, an anti-CD72 nanobody of the present invention comprises a CDR1, CDR2, and CDR3 of a variable domain sequence of any one of SEQ ID NOS: 1-8. In some embodiments, an anti-CD72 nanobody of the present invention comprises a CDR1, CDR2, and CDR3 of a variable domain sequence of any one of SEQ ID NOS:4, 5, or 6. In some embodiments, an anti-CD72 nanobody of the present invention comprises a CDR1, CDR2, and CDR3 of the variable domain sequence of SEQ ID NO:6.
In some embodiments, an anti-CD72 nanobody of the invention has at least one, at least two, or three CDRs of a variable domain sequence of any one of SEQ ID NOS:9-26. In some embodiments, an anti-CD72 nanobody of the present invention comprises a CDR3 selected from the CDR3 sequences of a variable domain sequence of any one of SEQ ID NOS:9-26.
In some embodiments, an anti-CD72 nanobody comprises a variable region that comprises a CDR3 of any one of SEQ ID NOS: 1, 2, 5, 6, 7, or 8 in which 1, 2, 3, or 4 amino acids are substituted, e.g., conservatively substituted. In some embodiments, an anti-CD72 nanobody comprises a variable region that comprises a CDR3 of SEQ ID NO:3 in which 1, 2, or 3 amino acids are substituted, e.g., conservatively substituted. In some embodiments, an anti-CD72 nanobody comprises a CDR3 of SEQ ID NO:4 in which 1 amino acid is substituted, e.g., conservatively substituted. In some embodiments, a single chain variable region further comprises a CDR1 of any one of SEQ ID NOS: 1 to 8 in which 1, 2, or 3, e.g., 1 or 2 amino acids, are substituted, e.g., conservatively substituted; and/or a CDR2 as shown in one of SEQ ID NOS: 1-8 in which 1, 2, 3, or 4 amino acids are substituted, e.g., conservatively substituted. In some embodiments, an anti-CD72 nanobody comprises a variable region that comprises: a CDR1 of SEQ ID NO:6, or a variant thereof in which 1 or 2 amino acids are substituted, e.g., conservatively substituted; a CDR2 of SEQ ID NO:6; or a variant thereof in which 1, 2, or 3 amino acids are substituted, e.g., conservatively substituted; and a CDR3 of SEQ ID NO:6, or a variant thereof in which 1, 2, or 3 amino acids are substituted, e.g., conservatively substituted.
In some embodiments, an anti-CD72 nanobody comprises a variable region that comprises a CDR3 of any one of SEQ ID NOS:9-26 in which 1, 2, or 3 amino acids are substituted, e.g., conservatively substituted. In some embodiments, a single chain variable region further comprises a CDR1 of any one of SEQ ID NOS:9 to 26 in which 1, 2, or 3, e.g., 1 or 2 amino acids, are substituted, e.g., conservatively substituted; and/or a CDR2 as shown in one of SEQ ID NOS:9-26 in which 1, 2, 3, or 4 amino acids are substituted, e.g., conservatively substituted.
In some embodiments, an anti-CD72 nanobody of the present invention comprises a single chain variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of a variable region sequence of any one of SEQ ID NOS:1-8. In some embodiments, the variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in any one of SEQ ID NOS: 1-8. In some embodiments, a nanobody of the present invention comprises an FR1-FR2-FR3-FR4 framework sequence that has at least 80% or at least 85% identity to the FR1-FR2-FR3-FR4 framework sequence of any one of SEQ ID NOS:1-8. 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 NOS: 1-8 from the N-terminus to the C-terminus without the three CDR sequences.
In some embodiments, an anti-CD72 nanobody of the present invention comprises a single chain variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of a variable region sequence of any one of SEQ ID NOS:9-26 In some embodiments, the variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in any one of SEQ ID NOS:9-26. In some embodiments, a nanobody of the present invention comprises an FR1-FR2-FR3-FR4 framework sequence that has at least 80% or at least 85% identity to the FR1-FR2-FR3-FR4 framework sequence of any one of SEQ ID NOS:9-26. 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 NOS:9-26 from the N-terminus to the C-terminus without the three CDR sequences.
In some embodiments, the FR1 region of a nanobody of the present invention comprises an FR1 sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to the FR1 sequence of any one of SEQ ID NOS:1-8. In some embodiments, the FR1 region of a nanobody of the present invention comprises an FR1 sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to the FR1 sequence of any one of SEQ ID NOS:9-26.
In some embodiments, the FR2 region of a nanobody of the present invention comprises an FR2 sequence having at least 80%, at least 85%, at least 95%, at least 90%, or at least 95% identity to the FR2 sequence of any one of SEQ ID NOS: 1-8. In some embodiments, the FR2 region of a nanobody of the present invention comprises an FR2 sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to the FR2 sequence of any one of SEQ ID NOS:9-26.
In some embodiments, the FR3 region of a nanobody of the present invention comprises an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to the FR3 sequence of any one of SEQ ID NOS:1-8. In some embodiments, the FR3 region of a nanobody of the present invention comprises an FR3 sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to the FR3 sequence of any one of
SEQ ID NOS:9-26.
In some embodiments, the FR4 region of a nanobody of the present invention comprises an FR4 sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to the FR4 sequence of any one of SEQ ID NOS: 1-8. In some embodiments, the FR4 region of a nanobody of the present invention comprises an FR4 sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to the FR4 sequence of any one of SEQ ID NOS:9-26.
As used herein with respect to the anti-CD72 nanobodies featured in the disclosure, “humanize” means that a nanobody VH sequence described herein is modified to produce a variant in which one or more amino acid residues are substituted to correspond to amino acid residues present at the corresponding positions of a human VH region, e.g., to reduce immunogenicity. In some embodiments, such changes are introduced into the framework region, e.g., one or more of FR1, FR2, FR3 or FR4. The term thus includes changing some, but not all, residues in an anti-CD72 nanobody sequence that differ from canonical human FR residues at the corresponding position.
The amino acid sequences of SEQ ID NOS: 1-26 differ from canonical human VH amino acid residues at positions that include, but are not limited to, positions 1, 5, 14, 35, 37, 44, 45, 47, 76, 80, 88, and 89 as determined with reference to any one of SEQ ID NOS: 1-8; or as determined with reference to SEQ ID NO:6. In some embodiments, an amino acid residue at one or more of the positions is substituted with the corresponding residue from a human sequences. In some embodiments, residues may be substituted to an alternative residue from a llama VH region sequences to provide improved properties such as solubility or stability.
In some embodiments, a nanobody comprising a CDR1, CDR2, and CDR3 as described herein, e.g., a nanobody comprising the CDR1, CDR2, and CDR3 of any one of SEQ ID NOS: 1-26, comprises an amino acid sequence in which at least one of positions 1, 5, 14, 35, 37, 44, 45, 47, 76, 80, 88, and 89 as determined with reference to any one of SEQ ID NOS:1-8 (or as determined with reference to SEQ ID NO:6) is substituted. In some embodiments, a variant comprises at least one amino acid residue selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89. In some embodiments, a variant comprises two amino acid residues selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89. In some embodiments, a variant comprises three amino acid residues selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89. In some embodiments, a variant comprises four amino acid residues selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89. In some embodiments, a variant comprises five amino acid residues selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89. In some embodiments, a variant comprises six residues selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89. In some embodiments, a variant comprises seven amino acids residue selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89. In some embodiments, a variant comprises eight amino acid residues selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89. In some embodiments, a variant comprises nine, ten, eleven, or all twelve amino acid residue selected from E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q or G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88 and A at position 89.
In preferred embodiments, FR4 comprises the sequence YWGQGTQVTVSS. In alternative embodiments, Q at position 7 of YWGQGTQVTVSS may be substituted. In some embodiments, L is substituted for Q at position 7 of YWGQGTQVTVSS. This position corresponds to position 117 of SEQ ID NO:6.
In some embodiments, a variant comprises Q at position 1, V at position 5, P at position 14, G at position 35, F at position 37, Q at position 44, R at position 45, L at position 47, A at position 76, V at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises Q at position 1, V at position 5, A at position 14, G at position 35, F at position 37, Q at position 44, R at position 45, L at position 47, A at position 76, V at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises Q at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, Q at position 44, R at position 45, L at position 47, A at position 76, V at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises Q at position 1, V at position 5, P at position 14, G at position 35, F at position 37, E at position 44, R at position 45, L at position 47, A at position 76, V at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises Q at position 1, V at position 5, P at position 14, G at position 35, F at position 37, Q at position 44, R at position 45, F at position 47, A at position 76, V at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, F at position 37, Q at position 44, R at position 45, L at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises Q at position 1, V at position 5, P at position 14, G at position 35, F at position 37, Q at position 44, R at position 45, L at position 47, A at position 76, V at position 80, R at position 88, and P at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises Q at position 1, V at position 5, A at position 14, G at position 35, F at position 37, Q at position 44, R at position 45, L at position 47, A at position 76, V at position 80, R at position 88, and P at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises Q at position 1, V at position 5, A at position 14, G at position 35, F at position 37, Q at position 44, R at position 45, F at position 47, A at position 76, V at position 80, R at position 88, and P at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises Q at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, Q at position 44, R at position 45, F at position 47, A at position 76, V at position 80, K at position 88, and P at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 47, A at position 76, V at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS:1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, Q at position 44, R at position 45, L at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, Y at position 37, Q at position 44, R at position 45, L at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, Y at position 37, G at position 44, L at position 45, L at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, E at position 44, R at position 45, F at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, E at position 44, R at position 45, F at position 47, S at position 76, V at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, G at position 44, R at position 45, F at position 47, S at position 76, L at position 80, R at position 88, and P at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, G at position 44, R at position 45, F at position 47, S at position 76, V at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, G at position 44, R at position 45, F at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, G at position 44, L at position 45, F at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, Y at position 37, G at position 44, L at position 45, F at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, Y at position 37, G at position 44, R at position 45, F at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, Y at position 37, E at position 44, R at position 45, F at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, Y at position 37, G at position 44, R at position 45, F at position 47, S at position 76, L at position 80, R at position 88, and P at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, Y at position 37, G at position 44, R at position 45, F at position 47, S at position 76, V at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, Y at position 37, G at position 44, R at position 45, L at position 47, S at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, Y at position 37, G at position 44, R at position 45, F at position 47, S at position 76, V at position 80, R at position 88, and P at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 47, S at position 76, L at position 80, K at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, G at position 44, R at position 45, F at position 47, S at position 76, L at position 80, K at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, G at position 44, R at position 45, F at position 47, A at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, G at position 35, Y at position 37, G at position 44, L at position 45, F at position 47, S at position 76, L at position 80, R at position 88, and P at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, Y at position 37, G at position 44, R at position 45, F at position 47, A at position 76, L at position 80, R at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
In some embodiments, a variant comprises E at position 1, V at position 5, P at position 14, S at position 35, Y at position 37, G at position 44, R at position 45, F at position 47, S at position 76, L at position 80, K at position 88, and A at position 89 as determined with reference to any one of SEQ ID NOS: 1-8 is substituted, for example, as determined with reference to SEQ ID NO:6.
A humanized nanobody as described in this section typically retains at least 50% activity compared to antibody NbD4 as assessed at a 1:1 effector:target ratio in a CAR T in vitro cytotoxicity assay against the SEM B-ALL cell line performed as described in Example 4.
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 the 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 comprises more than one anti-CD72 nanobody as described herein, e.g., two, three, or four anti-CD72 nanobodies of the present invention, e.g., where the nanobodies are joined by linkers.
In some embodiments, a nanobody of the present invention is linked to a second nanobody, e.g., a second anti-CD72 nanobody as described herein, 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 invention 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.
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).
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 (CD134). 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.
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 any one of SEQ ID NOS: 1-8, e.g., 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. In some embodiments, the anti-CD72 nanobody domain comprises a humanized version of any one of SEQ ID NOS:9-26, e.g., 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.
In some embodiments, the extracellular domain may comprise two more anti-CD72 nanobodies as described herein. 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, or other anti-CD72 antibody, that binds to a different CD72 epitope. In some embodiments at least one of the nanobodies comprises a CDR1 sequence comprising TISPIDI, a CDR2 sequence comprising FVAAIALGGN or LVAAIALGGN, 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.
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 immunglobuline 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.
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, OX40, CD2, CD27, LFA-1 (CD11a, 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, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, 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, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME, (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, or NKG2C.
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 or NK 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).
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.
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 R11a, FcR beta (Fc Epsilon Rib), CD3 gamma, 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.
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, 4-1BB (CD137), OX40, 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, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB 1, CD29, ITGB2, CD18, 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, Ly108), SLAM, (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, and CD 19a.
In some embodiments, a CAR may be designed as an inducible CAR, or may otherwise comprise a mechanisms for reversibly expressing the CAR, or controlling CAR activity to largely restrict it to a desired environment. Thus, for example, 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., 41BB), 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.
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.
In some embodiments, a cell expressing a CAR comprising one or more anti-CD72 nanobodies as described herein also 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 CD19, that is expressed on a B cell malignancy.
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.
Immune effector cells such as T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 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.
Methods of making CAR-expressing cells are described, e.g., in US2016/0185861 and US2019/0000880.
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. Non-limiting 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).
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 into immune effectors cells, which may then be selected and expanded for administration to a patient.
In a further aspect, an anti-CD72 nanbody of the present invention 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.
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, cc1065, 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.
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.
An anti-CD72 nanobody, an antigen binding molecule comprising an anti-CD72 nanobody, or an effector cell, e.g., T-cell, genetically modified a CAR 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.
In some embodiments, an anti-CD72 nanobody, an antigen binding molecule comprising an anti-CD72 nanobody, or an effector cell, e.g., T-cell, genetically modified a CAR 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-15Rα, 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-α, -β or -γ), erythropoietin and thrombopoietin, or a combination thereof. In some embodiments, the complex may be co-administered 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, β-glucan, an ISCOM, QS-21, or another immunopotentiating agent.
An anti-CD72 nanobody of the present invention, 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, 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, μ heavy-chain disease, γ heavy-chain disease, μ 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 11q 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 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. 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 as described herein is T-cell acute lymphoblastic leukemia, acute myeloid leukemia, or T-cell acute lymphoblastic leukemia.
In one aspect, a method of treating a B-cell malignancy 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. 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. 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.
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 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. 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.
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.
In some embodiments, pharmaceutical compositions of the present invention comprise a 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. In some embodiments, immune effector cells genetically modified to express a CAR comprising an anti-CD72 nanobody are formulated for intravenous administration.
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.
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 104 to 109 cells/kg body weight, in some instances 105 to 106 cells/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 3×104, 1×106, 3×106, or 1×107 cells/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 in to a particular site, such as a lymph node.
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.
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.
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.
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 (β-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.
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, 33:97
Administration in Combination with Other Agents
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 the second begins . . . . In some embodiment, treatment may be more effective because of combined administration.
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.
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.
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-norleucine, 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-11; 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.
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.
To define the B-ALL cell surfaceome, we enriched N-glycoproteins using a modified version of the Cell Surface Capture method (
We bioinformatically triaged our cell surface markers in order to identify potential immunotherapy candidates for MLLr B-ALL. We sequentially considered up-regulated cell surface markers in MLLr vs. other (63/799 proteins); relatively abundant proteins to find markers with high antigen density (LFQ intensity >25, 27/799); and single pass membrane proteins to facilitate development of in vitro antibodies (17/799) (
CD72, also known as lyb-2 in murine biology, is a single-pass Type-II membrane protein with an extracellular C-type lectin domain and cytoplasmic ITIM motifs. The ITIM motifs on CD72, similar to CD22, serve as scaffolds for inhibitory phosphatases to counteract B-cell receptor (BCR) signaling. These proteins, as well as CD19, demonstrate highly similar expression patterns across hematopoietic cell types per the HBA (
To independently verify these results, we examined CD72 surface expression on B-ALL patient-derived xenografts (PDX) from the ProXe biobank and viably frozen primary pediatric samples from our institution. By quantitative flow cytometry, we found CD72 to be expressed at several thousand copies per cell in MLLr PDX samples, similar to and sometimes greater than CD19 (
Altogether, these studies demonstrated that CD72 is highly restricted to the B-cell compartment, and highly abundant on not only MLLr leukemias, but also on many other B-cell malignancies including other B-ALL subtypes and lymphoma samples. Therefore, CD72 is an attractive surface receptor for targeting these B cell malignancies with new immunotherapy strategies to overcome emerging resistance mechanisms to CD19 and CD22 directed CAR-T therapy.
To generate CD72-specific binding reagents for use in a CAR-T cells, we employed a recently developed, fully in vitro nanobody yeast display screening platform (McMahon et al, Nat Struct Mol Biol. 1-14 (2018)) (
We expressed in mammalian cells a recombinant fusion protein comprised of the C-terminal extracellular domain of CD72 (aa 117-359) fused to a biotinylated human Fc domain to enable in vitro nanobody panning (
We cloned our unique nanobody sequences into a second-generation CAR-T format to screen activity in vitro. Notably, the lentiviral backbone (
To support CD72 CAR-T as either a front-line or second-line therapy after CD19 failure, we suppressed CD19 in SEM cells using CRISPRi to generate a model of CD19 antigen escape. CD72 (Nb.D4) CAR-T was equally efficacious against CD19-negative SEM cells as parental (
The in vivo efficacy of our CD72 CAR T was examined against an MLLr B-ALL cell line (SEM) and an MLLr B-ALL PDX in NOD scid gamma (NSG) mice. We engineered both cells to express luciferase for non-invasive bioluminescent imaging (BLI). 1e6 cells were implanted via tail vein injection and engraftment confirmed by BLI at either 3 or 10 days for SEM and PDX, respectively. Each cohort of mice (n=6 per arm) received 5e6 total CAR-T cells (a 1:1 mixture of CD4:CD8 primary T-cells) engineered with either an “empty” CAR backbone, CD72 (Nb.D4) CAR, or CD19 CAR. MLLr PDX-injected mice that received CD72 (Nb.D4) CAR-T showed a strong response and undetectable leukemic burden by BLI, comparable to CD19 CAR-T, and significantly increased survival versus the empty CAR (
The llama Nb framework sequence (FWR or FR), e.g., the framework sequence for antibody NbD4 (Nbd4 VH sequence SEQ ID NO:6), differs from canonical human VH FR residues by 13 amino acids, including residues Q1, Q5, and A14 in FR1; G35, Y37, E44, R45, and in FR2; A76, V80, K88, and P89 in FR3; and Q at position 7 of FR4 as shown in SEQ ID NO:6. The NbD4 sequence also differs at position 47 (i.e., position 1 of CDR2 as shown in SEQ ID NO:6) from the canonical human residue at the corresponding position. Due to the unique requirements for the nanobodies to not only bind CD72, but to also support CAR signaling, we exchanged llama FW residues with their human counterpart residues in different permutations, to determine combinations of residue changes that humanize the VH regions while retaining full CAR signaling efficacy. In addition to the noted FR residues, the NbD4 sequence at position 47 (position 1 of CDR2) was also varied as described below.
FR1 residues were exchanged in different combinations for consensus VH3 human residues, including introducing Q1->E1, Q5->V5, and A14->P14 substitutions.
FR2 llama residues, and in particular positions 37, 44, and 45; and position 47 (position 1 of CDR2), have been shown to play a role in the inherent solubility and folding of variable-heavy chain-only binding domains. We therefore adopted a combinatorial approach to change particular FR2 residues to determine how much modification is possible while retaining function. Llama residue G35 was either left unmodified or exchanged to S35, as a serine residue at this position is most common in human VH sequences. While position Y37 is typically V37 in humans, valine is not typically tolerated for retaining a stable VHH fold. We primarily retained the original llama residue Y37 but also utilized F37 in several variants, which is the residue present in some IGHV3 genes, and additionally utilized in the FDA-approved VHH Caplicizumab (Ablynx).
Llama residue E44 is typically G44 in humans. In addition to exchanging E44->G44, we also examined E44->Q44 to evaluate potential solubility compensation of glutamine in combination with other residue combinations. Residue R45 is typically L45 in humans, however leucine in this position has been shown to be detrimental to VHH solubility. Additionally, the original R45 is retained in the FDA-approved VHH
Caplicizumab, we therefore evaluated both R45 and L45 in our humanized variants. Position 47 is typically L47 in llama and W47 in humans. In NbD4 this position was F47, which although uncommon, is due to the synthetic nature of the original library design. W47 is not tolerated in VHH's, but we assessed retaining the original F47 as well as reverting to a more typical llama L47 and testing these different combinations for their effects on CAR-T efficacy, in combination with other humanized residue variants.
FR3 residues A76 and V80 were either retained or modified to standard VH3 residues S76 and L80. Similarly, residues K88 and P89 were either retained or changed to their human counterparts, R88 and A89.
Original llama FR4 residue Q117 as determined with reference to SEQ ID NO:6 was retained, as this residue was successfully utilized in the FDA-approved VHH Caplicizumab, demonstrated to possess low immunogenic potential.
CD8+ primary T cells were transduced with different CAR T constructs including CD19-CAR, CD72(NbD4)-CAR, and ten humanized variants of CD72(NbD4)-CAR (H1-H10). CAR T cells were co-cultured with the SEM B-ALL cell line (labeled with enhanced-firefly luciferase, effLuc) for 24 hours at different effector to target cell ratios. Target cell death was assessed using bioluminescence by adding D-luciferin to each well (final concentration 150 ug/ml) prior to luminescence imaging using a Promega GloMax Discover Microplate reader.
Humanized variants of CD72(NbD4) CAR-T (variants H3 and H10) were evaluated for their ability to degranulate in the presence of different B-cell malignancy cell lines, including SEM (MLLr B-ALL), RS411 (MLLr B-ALL), OCI-Ly18 (Diffuse-Large B-cell lymphoma, DLBCL), or Namalwa (Burkitt's Lymphoma) (
CD8+ primary T cells were transduced with CD72(NbD4) CAR-T and several humanized variants of CD72(NbD4)-CAR (H15, H20, H23, and H24 variants) utilizing an IgG4 hinge (EQ), CD28 costimulatory domain, and CD3 zeta receptor design (EQ28z).
To evaluate target cell killing of humanized NbD4 variants using the EQ28z receptor design, CAR T cells were co-cultured with the SEM B-ALL cell line (labeled with enhanced-firefly luciferase, effLuc) for 24 hours at different effector to target cell ratios. Target cell death was assessed using bioluminescence by adding D-luciferin to each well (final concentration 150 ug/ml) prior to luminescence imaging using a Promega GloMax Discover Microplate reader.
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.
LVAGIDTGANTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVAGINYGSNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVAGIAEGSNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVAGISYGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVAAIAAGGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
FVAAIALGGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
FVATIANGSNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVAGIGGGANTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVAGIDRGSNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVATISPGGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVATITQGATTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVAGISTGTITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVATIDPGGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVATITNGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVATISLGGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
ATISYGGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY
FVAAIDDGSITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
FVATIDVGGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVAGITYGANTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVAGIDLGGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVAGITSGTNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVASIADGTSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
LVAGISVGSNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
FVATITDGASTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
VAGIDVGAITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY
This application claims priority benefit to U.S. Provisional Application No. 63/134,427, filed Jan. 6, 2021, which is incorporated by reference for all purposes.
This invention was made with government support under grants no. K08 CA184116 and OD022552 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/011315 | 1/5/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63134427 | Jan 2021 | US |
