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  • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
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  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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Definitions

• the present invention encompasses the recognition that a combination of a cellular therapeutic described herein and one or more additional therapies (e.g., one or more additional cellular therapeutics (e.g., autologous CAR-T cell, allogenic CAR-T cell, CAR-NK cell, TCR-T cell, TIL cell, allogenic NK cell, autologous NK cell, gamma delta T cell, IPSC- derived cell, myeloid cell or other suitable cellular therapeutic cell type), antibody-drug conjugate, an antibody, and/or a polypeptide described herein), can lead to improved induction of beneficial immune responses, for example a cellular response (e.g., T-cell activation).
• additional therapies e.g., one or more additional cellular therapeutics (e.g., autologous CAR-T cell, allogenic CAR-T cell, CAR-NK cell, TCR-T cell, TIL cell, allogenic NK cell, autologous NK cell, gamma delta T cell, IPSC-
• FIG. 5D depicts exemplary CD 19 variants. Amino acids at the diversified positions are indicated for numerous clones.
• amino acid refers to any compound and/or substance that can be incorporated into a polypeptide chain.
• an amino acid has the general structure H2N-C(H)(R)-COOH.
• an amino acid is a naturally occurring amino acid.
• an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an 1-amino acid.
• Extracellular domain refers to a portion of a polypeptide that extends beyond the transmembrane domain into extracellular space.
• the term“gene” may include gene regulatory sequences (e.g ., promoters, enhancers, etc.) and/or intron sequences. It will further be appreciated that definitions of gene include references to nucleic acids that do not encode proteins but rather encode functional RNA molecules such as tRNAs, RNAi-inducing agents, etc.
• the term“gene” generally refers to a portion of a nucleic acid that encodes a protein; the term may optionally encompass regulatory sequences, as will be clear from context to those of ordinary skill in the art. This definition is not intended to exclude application of the term“gene” to non-protein coding expression units but rather to clarify that, in most cases, the term as used in this document refers to a protein-coding nucleic acid.
• expression control sequence refers to polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated.
• Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
• the nature of such control sequences differs depending upon the host organism.
• A“constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
• An“inducible” promoter is a nucleotide sequence that, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when a promoter-specific inducer is present in the cell.
• T cell receptor As used herein, a“T cell receptor” or“TCR” refers to the antigen-recognition molecules present on the surface of T-cells. During normal T-cell development, each of the four TCR genes, a, b, g, and d, can rearrange leading to highly diverse TCR proteins.
• regression of a particular tumor in an individual may also be assessed by taking samples of cancer cells from the site of a tumor such as a pancreatic adenocarcinoma (e.g., over the course of treatment) and testing the cancer cells for the level of metabolic and signaling markers to monitor the status of the cancer cells to verify at the molecular level the regression of the cancer cells to a less malignant phenotype.
• a tumor such as a pancreatic adenocarcinoma
• treatment refers to any administration of a substance that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition (e.g., cancer).
• a particular disease, disorder, and/or condition e.g., cancer
• Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
• such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
• Tumor infiltrating lymphocyte refers to white blood cells of a subject afflicted with a cancer (such as melanoma), that have left the blood stream and have migrated into a tumor.
• tumor-infiltrating lymphocytes have tumor specificity.
• the present disclosure provides cellular therapeutics, e.g., immune cells, genetically modified with an integrated gene, e.g., a nucleotide sequence encoding one or more biparatopic fusion proteins described herein (e.g., a constitutive expression construct and/or an inducible expression construct that includes such nucleotide sequence).
• a nucleotide sequence encoding a biparatopic fusion protein described herein can be designed to be constitutive or inducible by appropriate selection, construction and/or design of an expressed promoter sequence operably linked to such nucleotide sequence, as described herein.
• a gene in the construct is constitutively expressed.
• a signaling domain further includes one or more additional signaling regions (e.g., costimulatory signaling regions) that activate one or more immune cell effector functions (e.g., a native immune cell effector function described herein).
• additional signaling regions e.g., costimulatory signaling regions
• a portion of such costimulatory signaling regions can be used, as long as the portion transduces the effector function signal.
• a cytoplasmic domain described herein includes one or more cytoplasmic sequences of a T cell co-receptor (or fragment thereof).
• a polypeptide antigen that binds to one or more known antibody-drug conjugates can be included in a biparatopic fusion protein described herein.
• a biparatopic fusion protein described herein (or an expression construct (e.g., a constitutive expression construct or inducible expression construct) encoding one or more such polypeptide antigens), or a cellular therapeutic comprising such expression construct, is administered to a subject in combination with one or more of these (or other) known antibody-drug conjugates.
• an expression construct e.g., a constitutive expression construct or inducible expression construct
• a cellular therapeutic comprising such expression construct
• CD 19 is a 95 kDa type I transmembrane glycoprotein that is used as a biomarker of B cell development (Wang et ak, Exp. Hematok Oncol. 1 :36 (2012)). CD19 expression in lymphoma and leukemia has made it an effective therapeutic target, especially for chimeric antigen receptor (CAR) T cell therapy (Maude et al., Blood 125:4017-4024 (2015)).
• CAR chimeric antigen receptor
• CD 19 The extracellular region of CD 19 was hypothesized to contain two C2-like immunoglobulin domains (see, e.g., Wang et al., Exp. Hematol. Oncol. 1 :36 (2012); Tedder et al., Nat. Rev. Rheumatol. 5:572-577 (2009)). This is supported by homology modeling (Soding et al., Nucleic Acids Res. 33:244-248 (2005)) (see Figure 3). However, a recently published structure demonstrated that CD 19 does not include C2-like immunoglobulin domains (Teplyakov et al., Proteins 86:495-500 (2016)).
• the disclosure provides biparatopic fusion proteins that include an antibody that is or includes a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:203-225, wherein the portion lacks one or more of (i)-(v) (and/or lacks a portion of one or more of (i)-(v)).
• the disclosure provides biparatopic fusion proteins that include an antibody that is or includes a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:203-225, wherein the portion lacks one or more of the C-terminal amino acids
• any such CDR sequence may be readily combined, e.g., using molecular biology techniques, with any other antibody sequences or domains provided herein or otherwise known in the art, including any framework regions, CDRs, or constant domains, or portions thereof as disclosed herein or otherwise known in the art, as may be present in an antibody or binding molecule of any format as disclosed herein or otherwise known in the art.
• a tumor antigen described herein can be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).
• TSA is (or is believed to be) unique to tumor cells and does not occur on other cells in the body (e.g., does not occur to a significant extent on other cells).
• a TAA is not unique to a tumor cell and instead is also expressed on a normal cell (e.g., expressed under conditions that fail to induce a state of immunologic tolerance to the antigen).
• TAAs can be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they can be antigens that are normally present at extremely low levels on normal cells but that are expressed at higher levels on tumor cells.
• tumor antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, erbB, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72,
• a masked biparatopic fusion protein includes a masking moiety on one or more of the antigen binding proteins.
• a masking moiety is at the N-terminus of one or more antigen binding proteins included in an expressed biparatopic fusion protein.
• a masking moiety is at the C-terminus of one or more antigen binding proteins included in an expressed biparatopic fusion protein.
• a masked fusion protein described herein includes a linker, e.g., C-terminal and/or N-terminal to a masking moiety and/or cleavage moiety.
• a linker may provide flexibility for the masking moiety to reversibly inhibit binding of the antigen-binding protein to its target.
• Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.
• a masking moiety is fused to an antigen-binding protein through a polypeptide linker.
• a linker used to fuse a masking moiety to an antigen-binding protein is a cleavable moiety described herein.
• Recombinant expression vectors can be prepared using standard recombinant
• a recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the recombinant expression vectors include, for instance, neomycin/G418 resistance genes, puromycin resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes. [0155] Vectors useful in the context of the disclosure can be“naked” nucleic acid vectors
• Vector DNA or RNA can be introduced into a cell, e.g., an immune cell, via conventional transformation or transfection techniques.
• a cell e.g., an immune cell
• a number of expression vectors can be used, including, but not limited to, the E. coli expression vector pTIR278 (Ruther et al., 1983, EMBO 12: 1791); pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like.
• pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).
• monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et ah, Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
• a composition suitable for parenteral administration can be an aqueous or nonaqueous, isotonic sterile injection solution, which can contain anti-oxidants, buffers, bacteriostats, and solutes, for example, that render the composition isotonic with the blood of the intended recipient.
• An aqueous or nonaqueous sterile suspension can contain one or more suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
• Dosage administered to a subject, particularly a human, will vary with the particular embodiment, the composition employed, the method of administration, and the particular site and subject being treated. However, a dose should be sufficient to provide a therapeutic response.
• a clinician skilled in the art can determine the therapeutically effective amount of a composition to be administered to a human or other subject in order to treat or prevent a particular medical condition. The precise amount of the composition required to be therapeutically effective will depend upon numerous factors, e.g., such as the specific activity of the cellular therapeutic, and the route of administration, in addition to many subject-specific considerations, which are within those of skill in the art.
• Any suitable number cellular therapeutic cells can be administered to a subject.
• a dose of a cellular therapeutic described herein can be administered to a mammal at one time or in a series of subdoses administered over a suitable period of time, e.g., on a daily, semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, semi-annual, or annual basis, as needed.
• a dosage unit comprising an effective amount of a cellular therapeutic may be administered in a single daily dose, or the total daily dosage may be administered in two, three, four, or more divided doses administered daily, as needed.
• a tumor is or comprises a hematologic malignancy, including but not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, AIDS-related lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Langerhans cell histiocytosis, multiple myeloma, or myeloproliferative neoplasms.
• a tumor is a melanoma.
• a tumor is a B cell tumor.
• a cellular therapeutic and/or a protein therapeutic is administered in combination with a second cellular therapeutic, an antibody-drug conjugate, an antibody, and/or a polypeptide.
• the extent of tumor targeting and/or killing by a second cellular therapeutic is higher than a level observed or measured in the absence of combined therapy with a cellular therapeutic or a protein therapeutic described herein.
• ELISA ELISA.
• a 96-well ELISA plate was coated with 1 mg/ml of anti-CD 19 monoclonal antibody FMC63 in 0.1 M Carbonate, pH9.5. The plate was left to incubate overnight at 4°C. The coated plate was blocked with TBS/0.3% nonfat dry milk (NFD) for 60 minutes at RT. The plate was washed with TBST (0.1 M Tris, 0.5 M NaCl, 0.05% Tween 20. The purified biparatopic fusion protein was diluted in TBS/BSA and added at varying amounts from 0.005 mg/ml to 1 pg/ml, covering more than three logs of final concentration.
• Example 3 Cytotoxicity of Constitutively Expressed Biparatopic Constructs.
• sequences encoding a CD 19 binding CAR upstream of biparatopic fusion proteins were introduced to T cells by lentiviral transduction.
• the cells were washed with PBS, suspended in 50 m ⁇ FACS buffer (PBS +1% BSA and 0.1% sodium azide ) and blocked with human Fc block (Becton Dickinson) at room temperature for 10 minutes. Then, 50 pi of the fusion protein, #357 or #518, starting at 1 mg/ml with 3 fold dilutions in FACS buffer was added. The cell/fusion protein mixture was incubated for 30 minutes at 4 °C and then washed twice with FACS buffer. The cells were suspended in 100 m ⁇ of FACS buffer, stained with FMC63-PE (Millipore 5 m ⁇ /test), and incubated for 30 minutes at 4 °C. The cells were washed twice with FACS buffer, fixed with a final concentration of 1% PFA in PBS (Thermo Scientific) and then analyzed by flow cytometry.
• FACS buffer PBS +1% BSA and 0.1% sodium azide
• human Fc block Becton Dickinson
• the percent killing was calculated based upon the average loss of luminescence of the experimental vs the control (untreated) cells.
• Figure 16A shows the HIS-tagged biparatopic construct (#357) and the construct with no HIS-tag (#518) demonstrate equivalent binding by FACS on the CLEC12A-positive cell line AML5.
• a similar equivalence is observed using the CLEC12A-positive cell line U937 ( Figure 16B).
• Figure 16C The equivalent ability of both fusion proteins to bridge CAR-CD19 T cells to U937 target cells and mediate potent cytotoxicity is demonstrated in Figure 16C.
• U937 (ATCC), PL-21 and OCI-AML-5 (DSMZ) cells were cultured as detailed by the supplier. Luciferase expressing lines were generated using a lentivirus (GeneCopoeia) and puromycin selection. Cells carrying the luciferase gene were seeded at 1 c 10 4 cells in 50 pi per well in a 96 well round bottom plate in RPMI 1640 + 10% FBS without antibiotics. CAR T cells were thawed and washed once with RPMI/FBS via centrifugation at 450 RCF for 10 minutes.
• Biparatopic fusion protein concentration was determined in the CAR-405 expansion culture medium (batch fusion protein) as well as through stimulation post-freezing (fusion protein secretion).
• fusion protein secretion For measurement of biparatopic fusion protein during expansion at the time of harvest (day 10), 0.5ml of cell-free culture media was collected and frozen at -20°C.
• CAR-405 T cells were thawed and resuspended at 3 x 10 6 cells/ml in RPMI-1640/10% FBS.
• mice [0230] To confirm the in vivo efficacy of CAR-CD19 T cells that secrete biparatopic fusion proteins were introduced into NSGTM mice. All animal studies were performed in accordance with Tufts University IACUC approved guidelines. 6-8 week old NSGTM mice (Jackson Laboratories, NOD.Cg -Prkdc scid Il2rgf mlWjl l SzJ) were used. Mice were inoculated with 25,000 U937-luciferase cells IV. On day 3, 10 7 CAR-405, CAR-468 or untransduced T cells (UTD) from donors 38 or 45, were injected IV into the mice; a cohort of mice had no T cell injection (NA).
• fusion proteins #186, #330 or #357 were added.
• the cell/fusion protein mixture was incubated for 30 minutes at 4 °C and then washed twice with FACS buffer.
• the cells were suspended in 100 pi of FACS buffer, stained with HIB19-PE (BioLegend 5 m ⁇ /test), and incubated for 30 minutes at 4 °C.
• the cells were washed twice with FACS buffer, fixed with a final concentration of 1% PFA in PBS (Thermo Scientific) and then analyzed by flow cytometry.
• Table 10 provides ELISA results demonstrating the binding capacity of the dual antigen binding fusion protein (#440); the anti-CLEC12A VHH 2H3 fusion protein (#330); and the anti-CD33 scFv fusion protein (#410) to CLEC12A and CD33.
• the dual antigen binding fusion protein binds equally well to both components (CLEC12A and CD33) with similar EC so values showing that each individual component in the dual antigen construct is acting independently.
• Figure 20 demonstrates that the dual antigen binding fusion protein is capable of binding to U937 cells expressing each individual antigen.
• Figure 21 shows the cytotoxicity of the three fusion proteins on U937 (Fig. 21B) and Molml4 (Fig. 21 A) cell lines.

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