Compositions that target CD47 and CD138 and methods of making and using the same

Abstract

Disclosed are bispecific single-chain constructs comprising an anti-CD47 domain and an anti-CD138 domain, pharmaceutical compositions comprising the constructs, methods of treatment using the pharmaceutical compositions, polynucleotides encoding the constructs, methods of making the constructs, and kits. In some embodiments, the anti-CD47 domain comprises a first heavy chain and a first light chain and the anti-CD138 domain comprises a second heavy chain and a second light chain.

Description

Atty. Dkt. No. 650053.01238COMPOSITIONS THAT TARGET CD47 AND CD138 AND METHODS OF MAKING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 715,1 15 that was filed November 1 , 2024, the entire contents of which are hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.SEQUENCE LISTING

[0003] A Sequence Listing accompanies this application and is submitted as an xml file of the sequence listing named “650053_01238.xml” which is 72,792 bytes in size and was created on October 30. 2025. The sequence listing is electronically submitted via Patent Center and is incorporated by reference herein in its entirety.BACKGROUND

[0004] CD47 is a “do not eat me signal” that is overexpressed on malignant tumors including multiple myeloma (MM). CD47 on tumor cells interacts with SIRP-a on macrophages and inhibits its phagocytosis enabling tumor progression. Clinical studies with antibodies blocking CD47-SIRP-a interaction to promote tumor phagocytosis have shown varying success. CD47 is widely expressed on normal tissue and their binding to these monoclonal antibodies can lead to loss of treatment efficacy due to “sink effect” and to off-tumor on-target toxicity . In addition, when used as an adjuvant, CD47 antibody had a deleterious effect on CAR T cell survival and efficacy (Y amada-Hunter et al., Nature 2024). Accordingly, improved agents that target CD47 are needed in the art and are needed for the treatment of cancer, e.g., multiple myeloma.SUMMARY

[0005] In an aspect of the current disclosure, bispecific single-chain polypeptide constructs are provided. In some embodiments, the anti-CD47 domain comprises a first heavy chain and a first light chain and the anti-CD138 domain comprises a second heavy chain and a second light chain.Atty. Dkt. No. 650053.01238

[0006] In an aspect of the current disclosure, polynucleotides are provided. In some embodiments, the polynucleotides comprise a sequence encoding a bispecific single-chain polypeptide construct comprising an anti-CD47 domain and an anti-CD138 domain.

[0007] In an aspect of the current disclosure, cells comprising a polynucleotide comprising a sequence encoding a bispecific single-chain polypeptide construct comprising an anti-CD47 domain and an anti-CD138 domain.

[0008] In an aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise a bispecific single-chain polypeptide construct comprising an anti-CD47 domain and an anti-CD138 domain, optionally, further comprising a suitable excipient.

[0009] In some embodiments, the pharmaceutical compositions comprise a cell comprising a polynucleotide comprising a sequence encoding a bispecific single-chain polypeptide construct comprising an anti-CD47 domain and an anti-CD138 domain.

[0010] In an aspect of the current disclosure, methods are provided. In some embodiments, the methods comprise contacting a bispecific single-chain polypeptide construct comprising an anti-CD47 domain and an anti-CD138 domain to a cell.

[0011] In some embodiments, the methods comprise administering a pharmaceutical composition comprising a cell comprising a polynucleotide comprising a sequence encoding a bispecific single-chain polypeptide construct comprising an anti-CD47 domain and an antiCD 138 domain to a subject.

[0012] In some embodiments, the methods are methods of treating a disease or disorder associated with CD47 expression in a subject in need thereof and the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a bispecific single-chain polypeptide construct comprising an anti-CD47 domain and an antiCD 138 domain to the subject to treat the disease or disorder associated with CD47 expression in the subject.

[0013] In an aspect of the current disclosure, methods of treating a cell proliferative disease or disorder in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a bispecific single-chain polypeptide construct comprising an anti-CD47 domain and an anti-CD138 domain to the subject to treat the cell proliferative disease or disorder.

[0014] In an aspect of the current disclosure, further methods of treating a cell proliferative disease or disorder in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceuticalAtty. Dkt. No. 650053.01238 composition comprising a bispecific single-chain polypeptide construct comprising an anti- CD47 domain and an anti-CD138 domain and a chimeric antigen receptor (CAR) T cell therapy to the subject to treat the cell proliferative disease or disorder in the subject.

[0015] In an aspect of the current disclosure, further methods of treating a cell proliferative disease or disorder in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising cells expressing a construct comprising an anti-CD47 domain and an anti-CD138 domain.

[0016] In an aspect of the current disclosure, methods of making a CAR T cell that expresses a bispecific single chain construct are provided. In some embodiments, the methods comprise contacting a T cell with a polynucleotide comprising a sequence encoding a bispecific singlechain polypeptide construct comprising an anti-CD47 domain and an anti-CD138 domain and a CAR sequence.BRIEF DESCRIPTION OF THE FIGURES

[0017] FIG. 1 shows CD47-CD138 bispecific antibody (BsAb) expression in different formats. Antibodies were generated by transient expression using the Expi293 mammalian expression system. The antibodies have a His tag on the c terminal end which was used to pull down using Ni-NTA beads and eluted. The flow through (F.T) and the eluate for each antibody were run on 4-12% Bis-TRIS gel and stained with Coomasie blue.

[0018] FIG. 2 shows CD47-CD138 BsAb construct binds simultaneous to their corresponding antigens. A. schema for bridging ELISA. Recombinant CD138 was immobilized on maxisorp Nunc plates overnight and then incubated with PBS or CD47 scFv or CD47-CD138 (BsAb). After washing, biotinylated CD47 was added to the plate and incubated, washed and binding was detected by HRP-streptavidin. OD measured at 450 nm.

[0019] FIG. 3 shows binding of CD47-CD138 BsAb construct to myeloma cell line OPM2. CD47 scFv or CD47-CD138 BsAb have a FLAG tag at the C terminal end. Antibodies were incubated with OPM2 cells and then binding was detected by staining for FLAG using flow cytometry. Results shown in triplicate.

[0020] FIGs. 4A and 4B show CD47-CD138 BsAb construct increases phagocytosis of myeloma cells. OPM2 myeloma cell lines were labelled with CFSE and then incubated with macrophages in the presence of PBS or CD47 scFv or CD47-CD138 BsAb for 4 hours and phagocytosis was measured as percentage of CDl lb positive macrophages with CFSE using flow cytometry. Experiments performed in duplicates.Atty. Dkt. No. 650053.01238

[0021] FIG. 5 shows preferential binding of CD47-CD138 BsAb construct to myeloma cells in the presence of T cells. 0PM2 myeloma cells and primary T cells were mixed 1: 1 ratio and incubated with either CD47 scFv or CD47-CD138 BsAb and binding was measured using anti- FLAG antibody by flow cytometry. Samples assayed in triplicates (for each panel, the upper 3 histograms represent CD47 scFv binding and the lower 3 represent CD47-CD138 BsAb binding).

[0022] FIGs. 6A and 6B show CD47-CD138 BsAb construct mediates preferential phagocytosis of myeloma cells and relatively spares T cells. CFSE stained OPM2 cells and cell trace far red (CTFR) stained T cells were mixed in equal numbers with macrophages in the presence of PBS or CD47 scFv or CD47-CD138 BsAb and phagocytosis was assessed by the presence of either CFSE or CTFR in CD 11b positive macrophages by flow cytometry’, experiments performed in duplicate.

[0023] FIG. 7 shows CD47-CD138 BsAb treatment improves tumor control in a xenograft model of multiple myeloma. 6-7-week-old female NSG mice were implanted with luciferase expressing OPM2 cells subcutaneously. After confirming engraftment of the tumor by IVIS, mice were treated with either PBS or CD47 scFv or CD47-CD138 BsAb daily intraperitoneally for 4 weeks. Tumor progression measured by luciferase activity using IVIS imaging.

[0024] FIGs. 8A, 8B, and 8C show Production of antibody variants. (A). LX293 cells transfected with expression constructs that generate bispecific antibodies with different linkers. (B) Expi293 cells transfected with expression constructs that generates CD47 scFv or CD47- CD138 BsAb with or without Fc. (1C) Expi293 cells transfected to express CD47scFv, CD138 scFv and CD47-CD138 with HSA linker BsAb without Fc domain.

[0025] FIG. 9 shows TRF binding assay. Solid-phase binding of CD47 scFv and CD47- CD138 BsAb antibodies to recombinant human CD47 and CD138 as determined by time- resolved fluorescence (TRF) assay. TRF raw signals determined on Tecan plate reader.

[0026] FIG. 10 shows Bridging ELISA assay. CD47-CD138 BsAb simultaneous binding to both CD47 and CD138 cognate antigens determined by bridging ELISA assay. Bridging ELISA raw data determined on Tecan plate reader.

[0027] FIG. 11 shows CD47-CD138 BsAb binding over time. Purified CD47-CD138 BsAb (1.3uM) was incubated with OPM2 MM cell line followed by anti-FLAG-tag staining at different time points using flow cytometry' (median fluorescence intensity (MFI) 1 month: 1.25E6, MFI 2 months: 1.29E6, MFI 3 months: 1.32E6 and MFI 1 year: 1.20E6).Atty. Dkt. No. 650053.01238

[0028] FIG. 12 shows cell-based binding assay with commercial antibodies. OPM2 and MM1S myeloma cell lines were stained with human anti-CD138-APC, anti-CD47-APC or anti-BCMA-APC commercial antibodies.

[0029] FIGs. 13A and 13B show cell-based binding assay with in-house produced antibodies. Equimolar concentrations (1.3 uM) of CD47-CD138 BsAb, CD138 scFv or CD47 scFv were incubated with OPM2 or MM IS MM cell line followed by anti-FLAG-tag staining. (A) Histogram showing binding of these antibodies to different cell lines. (B) Median fluorescence intensity of binding of CD47-CD138 BsAb (HSA linker) and CD47 scFv to OPM2 cells by flowcylometry (n=3).

[0030] FIG. 14 shows T-cell binding assay. Equimolar concentrations (1.3 uM) of CD47- CD138 BsAb or CD47 scFv were incubated with isolated human T-cells followed by anti- FLAG-tag staining. Representative histogram and data compiled of binding of CD47-CD138 BsAb (HSA linker) and CD47 scFv to OPM2 cells analyzed by flowcylometry (n=3).

[0031] FIG. 15 shows flow cytometry preferential binding assay. U266 cell line was mixed with equal number of T-cells in the presence of O.OluM of CD47 scFv or CD47-CD138 BsAb and stained with anti-FLAG-tag (APC) and CD3 (BUV395). Median fluorescence intensity (MFI) of anti-FLAG-tag antibody representing CD47 scFv or CD47-CD138 BsAb binding on co-cultured T-cells and U266 cell line.

[0032] FIGs. 16A and 16B show in-vitro phagocytosis assay. A. Normalized phagocytosis of OPM2 multiple myeloma cell line (n = 4) or B T-cells (n=4) in the presence of equimolar concentrations (1 .3 uM) of CD47-CD138 BsAb or CD47 scFv and PBS control.

[0033] FIGs. 17A and 17B show in-vitro preferential phagocytosis assay. Normalized phagocytosis of OPM2 MM cell line (A) vs T cells (B) (n = 4) in a mixed coculture with a E:T ratio of I :0.5:0.5 (macrophages: OP M2 MM cells:T cells) in the presence of equimolar concentrations (1.3 uM) of CD47-CD138 BsAb or CD47 scFv and PBS control.

[0034] FIG. 18 shows generation of OPM2-BCMAKOand U266-BCMAKOby CRISPR- CAS9. Wildty pe MM cells and BCMAKOcells (after sorting) were stained with human anti- BCMA (APC) to confirm BCMAKOcells and assessed by flow cytometry’.

[0035] FIG. 19 shows phagocytosis of myeloma cells lacking BCMA expression. OPM2 and OPM2 BCMAK(Jcells (labelled with CTFR) were incubated with BCMA CAR T cells in the presence of macrophages and CD47 scFv or CD47-CD138 BsAb or PBS control for 4 hours and phagocytosis determined by flow cytometry.

[0036] FIGs. 20A and 20B show secreted proteins from CAR T cells supernatant as determined by western blot. (A) Internal controls. Lane 1 = spiked supernatant sample withAtty. Dkt. No. 650053.01238 purified CD47 scFv, Lane 2 = spiked supernatant sample with purified CD47-CD138 BsAb, Lane 3 = purified CD47 scFv, Lane 4 = purified CD47-CD138 BsAb. (B) Lane 1 = BCMA CAR T cell secrets CD47 scFv, Lane 2 = BCMA CAR T cell secrets CD47-CD138 BsAb, Lane 3 = BCMA CAR T cell.DETAILED DESCRIPTION

[0037] Disclosed herein are bispecific single-chain polypeptide constructs comprising an anti-CD47 domain and an anti-CD138 domain, polypeptides encoding the bispecific singlechain polypeptide constructs, cells comprising the polypeptides, pharmaceutical compositions comprising the bispecific single-chain polypeptide constructs or the cells, methods of treating subjects using the disclosed pharmaceutical compositions, methods of making the bispecific single-chain polypeptide constructs, methods of making chimeric antigen receptor (CAR) T cells expressing the bispecific single-chain polypeptide constructs, and kits comprising the bispecific single-chain polypeptide constructs.Bispecific single-chain polypeptide constructs

[0038] CD47 is associated with several malignancies including multiple myeloma (MM). However, CD47 is widely expressed on normal tissue and their binding to these monoclonal antibodies can lead to loss of treatment efficacy due to “sink effect” and to off-tumor on-target toxicity. To overcome these limitations and improve the on-tumor effect of CD47 blockade in MM, the inventors created a bispecific single-chain polypeptide construct comprising an anti- CD47 single chain variable fragment (scFv) linked to an anti-CD138 scFv (FIG. 2). Since CD138 expression is mostly limited to MM the inventors hypothesized that CD47-CD138 construct will preferentially bind to MM cells, thereby improving anti-tumor efficacy and at the same time minimizing off-tumor toxicity.

[0039] In some embodiments, disclosed constructs comprise scFvs to target CD47, e.g., SEQ ID NO: 1 and CD138. e.g., SEQ ID NO: 4, and a linker connecting the scFvs, e.g.. SEQ ID NO: 14.

[0040] Accordingly, in an aspect of the current disclosure, bispecific single-chain constructs are provided. In some embodiments, the constructs comprise an anti-CD47 domain and an anti- CD138 domain. In some embodiments, the anti-CD47 domain and the anti-CD138 domain are antibody single chain variable fragments (scFvs) which are made up of an antibody heavy chain and light chain linked together. Thus, in some embodiments, the anti-CD47 domainAtty. Dkt. No. 650053.01238 comprises a first heavy chain and a first light chain, and the anti-CD138 domain comprises a second heavy’ chain and a second light chain.

[0041] In some embodiments, the first heavy chain (anti-CD47) comprises SEQ ID NO: 2, or a sequence with at least about 50% identity', at least about 55% identity7, at least about 60% identity , at least about 65% identity7, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity7, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity7, at least about 95% identity, at least about 96% identity, at least about 97% identity7, at least about 98% identity, or at least about 99% identity7to SEQ ID NO: 2.

[0042] In some embodiments, the first light chain (anti-CD47) comprises SEQ ID NO: 3, or a sequence with at least about 50% identity7, at least about 55% identity, at least about 60% identity7, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity7, at least about 90% identity7, at least about 91% identity, at least about 92% identity7, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 3.

[0043] In some embodiments, the second heavy chain (anti-CD138) comprises SEQ ID NO: 5, or a sequence with at least about 50% identity7, at least about 55% identity7, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity7, at least about 94% identity7, at least about 95% identity7, at least about 96% identity7, at least about 97% identity , at least about 98% identity, or at least about 99% identity7to SEQ ID NO: 5.

[0044] In some embodiments, the second light chain (anti-CD138) comprises SEQ ID NO: 6, or a sequence with at least about 50% identity, at least about 55% identity, at least about 60% identity7, at least about 65% identity, at least about 70% identity, at least about 75% identity7, at least about 80% identity7, at least about 85% identity, at least about 90% identity7, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 6.

[0045] In some embodiments, the anti-CD47 domain comprises the sequence SEQ ID NO: 1, or a sequence with at least about 50% identity, at least about 55% identity7, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity7, at least aboutAtty. Dkt. No. 650053.0123891% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 1.

[0046] In some embodiments, the anti-CD138 domain comprises SEQ ID NO: 4, or a sequence with at least about 50% identity7, at least about 55% identity, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity7, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 4.

[0047] In some embodiments, the constructs comprise a linker. In some embodiments, the linker is derived from human albumin, e.g., amino acids 427-451, e.g., a linker with the amino acid sequence SEQ ID NO: 14 or a sequence with 50% identity, at least about 55% identity, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 14. The linker may also comprise one of SEQ ID NOs: 17-19, or a sequence with at least about 50% identity, at least about 55% identity, at least about 60% identity, at least about 65% identity7, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to one of SEQ ID NOs: 17-19.

[0048] The lack of an antibody Fc domain may also improve the properties of the disclosed constructs by, for example, preventing ADCC towards tumor infiltrating lymphocytes expressing CD47. Accordingly, in some embodiments, the disclosed constructs do not comprise an antibody Fc domain.

[0049] In some embodiments, the directionality7of the construct may be defined. Accordingly, in some embodiments, the construct comprises, from N to C terminus, an anti-CD47 domain, a linker, and an anti-CD138 domain. In other embodiments, the construct comprises, from N to C terminus, an anti-CD138 domain, a linker, and an anti-CD47 domain. In some embodiments, the construct comprises one of SEQ ID NOs: 7-9 or a sequence with at leastAtty. Dkt. No. 650053.01238 about 50% identity, at least about 55% identity, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to one of SEQ ID NOs: 7-9.Polynucleotides

[0050] Also contemplated in the current disclosure are polynucleotides that encode the disclosed constructs. Accordingly, in another aspect of the current disclosure, polynucleotides are provided. In some embodiments, the polynucleotides comprise a sequence encoding a bispecific single-chain construct comprising an anti-CD47 domain and an anti-CD138 domain. The polynucleotides may be RNA molecules or DNA molecules. The polynucleotides may comprise or consist of one of SEQ ID NOs: 10-13, 15, or 16 or a sequence with at least about 50% identity, at least about 55% identity, at least about 60% identity, at least about 65% identity', at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity7, at least about 91% identity', at least about 92% identity, at least about 93% identity', at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity’, or at least about 99% identity to one of SEQ ID NOs: 10-13, 15, or 16. One of skill in the art would understand that certain modifications to the DNA sequence encoding the disclosed constructs, or portions thereof, could be modified to generate constructs with essentially the same amino acid structure, for example, through codon optimization or conservative codon usage.

[0051] The sequences encoding a bispecific single-chain construct may be operably linked to a regulatory element, e.g., a promoter or enhancer.

[0052] As used herein, “operably linked” refers to a functional linkage between two or more sequences such that activity at or on one sequence affects activity at or on the other sequence(s). For example, an operable linkage between a polynucleotide of interest, e.g., a sequence encoding a bispecific single-chain polypeptide construct of the instant disclosure, and a regulatory element (e.g., a promoter) is a functional link that allows for expression of the polynucleotide of interest.

[0053] The polynucleotides may further comprise regulatory sequences for use with vectors, e.g., long terminal repeats (LTRs) for use with retroviral vectors, e.g., lentiviral vectors.Atty. Dkt. No. 650053.01238

[0054] The polynucleotides may comprise at least one modified nucleotide. As used herein, “modified nucleotides” refer to nucleotide bases which are either not found in nature or are not proteinogenic nucleotides. Exemplary modified nucleotides may include, but are not limited to, modified nucleotides such as 2'-O-methyl (2'OMe) nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxy nucleotides, 2'-O-(2 -methoxy ethyl) (MOE) nucleotides, 1-methyl- pseudouridine (Mli ) and the like.Pharmaceutical compositions

[0055] The disclosed bispecific constructs are contemplated to be used in the treatment of diseases or disorders. Thus, in another aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise a bispecific single-chain construct comprising an anti-CD47 domain and an anti-CD138 domain. As will be understood by one of skill in the art, the disclosed pharmaceutical compositions may be formulated such that they include appropriate pharmaceutical carriers or excipients to maximize the desired pharmaceutical composition’s function. Production of such pharmaceutical formulations are considered routine in the art.

[0056] In some embodiments, the pharmaceutical compositions are formulated for parenteral administration, i.e., they may be formulated for administration by subcutaneous (SC / SQ), intraperitoneal (IP), intravenous (IV), intradermal (ID), and intramuscular (IM) route. Suitably, the pharmaceutical compositions are formulated for intravenous administration. In some embodiments, the pharmaceutical compositions are formulated such that they consist only of GMP approved compounds, i.e., they have defined characteristics, and may be used in conjunction with GMP prepared cells, e.g., the compositions may be used in conjunction with cells intended for adoptive transfer therapy.

[0057] The inert ingredients and manner of formulation of the pharmaceutical compositions are conventional. The usual methods of formulation used in pharmaceutical science may be used here. In general, compositions contain from about 0.5% to about 50% of the compound in total, depending on the desired doses and the type of composition to be used. The amount of the compound may be defined as the “effective amount”, that is, the amount of the compound which provides the desired dose to the patient in need of such treatment. The activity of the compounds employed in the compositions and methods disclosed herein are not believed to depend greatly on the nature of the composition, and. therefore, the compositions can be chosen and formulated primarily or solely for convenience and economy.Atty. Dkt. No. 650053.01238Methods of treatment

[0058] In another aspect of the current disclosure, methods of treating a disease or disorder associated with CD47 expression are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a bispecific single-chain construct comprising an anti-CD47 domain and an anti-CD138 domain to a subject in need thereof to treat the disease or disorder. As discussed above, CD47 is expressed on a variety of transformed cells, e.g., transformed cells present in a subject with a cell-proliferative disease or disorder. In some embodiments, the cell proliferative disease or disorder is a cancer, e.g., multiple myeloma; however, it is to be understood that the diseases and disorders associated with CD47 expression are not limited to the foregoing examples. Accordingly, cancers that are characterized by CD47 expression or CD 138 expression on the surface of cancerous cells are also contemplated to be targets for treatment by administration of the disclosed compositions, pharmaceutical compositions and by use of the disclosed methods.

[0059] The disclosed compositions preferentially bind to transformed / tumor cells rather than T cells (FIGs. 5 and 15). Therefore, in an aspect of this disclosure, methods of treating a cell proliferative disease or disorder in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of the disclosed pharmaceutical compositions and a chimeric antigen receptor (CAR) T cell therapy to the subject to treat the cell proliferative disease or disorder in the subject.

[0060] Several CAR T cell therapies are known in the art and are suitable for use in combination with the disclosed single chain constructs including, but not limited to: axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, tisagenlecleucel, ciltacabtagene autoleucel, and idecabtagene vicleucel.

[0061] The disclosed methods may comprise administering a therapeutically effective amount of a CAR T cell expressing the disclosed bispecific single chain polypeptide constructs to a subject. The cells, e.g., CAR T cells may express the disclosed constructs and secrete them extracellularly (FIG. 20B).

[0062] As used herein, "administration ' may comprise any acceptable administrative route, e.g., intravenous administration or intrathecal administration. Alternatively, administration may comprise administration by percutaneous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subject and the caregiver, etc.Atty. Dkt. No. 650053.01238

[0063] In some embodiments, the disclosed compositions, e.g., bispecific single chain constructs, can be directly administered to a subject in need thereof (e.g., a subject diagnosed with or suffering from a CD47 related disease or condition, such as multiple myeloma). By way of example, but not by w ay of limitation, administration of the bispecific antibody may include intravenous, intrathecal, intracranial, or intratumoral administration.

[0064] As used herein, a "‘therapeutically effective amount” refers to the amount of the administered composition that is effective in improving at least one sign or symptom in the subject, e g., at least one sign or symptom of a cell proliferative disease, e.g., reduction in tumor size, reduction in tumor burden, reduction in tumor grade or severity, or improvement in disease prognosis.Cells

[0065] The instant disclosure provides polynucleotides that encode the disclosed novel bispecific single-chain constructs. Accordingly, in another aspect of the current disclosure, cells comprising the disclosed polynucleotides encoding the bispecific single-chain constructs are provided. In some embodiments, the cells are a mammalian cells, e.g., human cells. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are T cells, e.g., CAR T cells, see, e.g., FIG. 20B.Methods of making the bispecific single-chain polypeptide constructs

[0066] In addition to the cells provided by the instant disclosure, methods of making the disclosed bispecific single-chain polypeptide constructs are provided. In some embodiments, the methods comprise introducing the disclosed polynucleotides into a cell and allowing the cell to express the polynucleotides to generate the disclosed bispecific single chain polypeptide constructs in vitro. In some embodiments, the constructs are further purified from the cells by means known in the art. In some embodiments, the constructs comprise affinity tags, e.g., streptavidin tags, histidine tags, FLAG tags, HA tags, etc., which allow their efficient purification.Methods of making chimeric antigen receptor T cells expressing the bispecific single-chain polypeptide constructs

[0067] In an aspect of the current disclosure, methods of making a CAR T cell that expresses a bispecific single chain construct are provided. In some embodiments, the methods comprise contacting a T cell with the disclosed polynucleotides wherein the polynucleotides furtherAtty. Dkt. No. 650053.01238 comprise a chimeric antigen receptor (CAR) sequence. In some embodiments, the methods comprise contacting a T cell with the disclosed polynucleotides and further contacting the T cell with a polynucleotide comprising a chimeric antigen receptor (CAR) sequence. The CAR T cells may be manufactured by either introducing into the cells a polynucleotide encoding the CAR and the disclosed constructs, or by introducing into the cells a first polynucleotide encoding the CAR and a second polynucleotide encoding the disclosed constructs.

[0068] SEQ ID NO: 15 is a polynucleotide encoding an anti-BCMA CAR and one embodiment of the disclosed constructs (SFG-BCMA-CAR-CD47-HSA CD138 BsAb-GFP vector).

[0069] SEQ ID NO: 16 is a polynucleotide encoding an anti-BMCA CAR.

[0070] The difference between SEQ ID NOs: 15 and 16 is that in SEQ ID NO: 15 the CD47- HSA-CD138 BsAb is encoded in the same plasmid construct as the BCMA CAR and in SEQ ID NO: 16 the CD47-HSA-CD138 BsAb is encoded in a separate plasmid.Kits

[0071] In an aspect of the current disclosure, kits are provided. In some embodiments, the kits comprise the disclosed bispecific single-chain polypeptide constructs and / or polynucleotides comprising the same. The kits may further comprise cells capable of expressing the bispecific single-chain polypeptide constructs, e.g., human cells, e.g., HEK cells.Further Definitions

[0072] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology7used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0073] As used in this specification and the claims, the singular forms “a,” "an." and "the" include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent’’ should be interpreted to mean ‘'one or more substituents,” unless the context clearly dictates otherwise.

[0074] As used herein, "‘about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.Atty. Dkt. No. 650053.01238

[0075] As used herein, the terms "include" and “including"’ have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being "‘open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0076] The phrase “such as"’ should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0077] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0078] All language such as “up to.” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3. 4, or 6 members, and so forth.

[0079] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or featureAtty. Dkt. No. 650053.01238 contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”Illustrative embodiments1. A bispecific single-chain polypeptide construct comprising an anti-CD47 domain and an anti-CD138 domain.2. The construct of embodiment 1 , wherein the anti-CD47 domain comprises a first heavy chain and a first light chain and the anti-CD138 domain comprises a second heavy chain and a second light chain.3. The construct of embodiment 1 or 2, wherein the anti-CD47 domain comprises SEQ ID NO: 1.4. The construct of any one of the preceding embodiments, wherein the anti-CD138 domain comprises SEQ ID NO: 4.5. The construct of any one of the preceding embodiments, wherein the first heavy chain comprises SEQ ID NO: 2.6. The construct of any one of the preceding embodiments, wherein the first light chain comprises SEQ ID NO: 3.7. The construct of any one of the preceding embodiments, wherein the second heavy chain comprises SEQ ID NO: 5.8. The construct of any one of the preceding embodiments, wherein the second light chain comprises SEQ ID NO: 6.9. The construct of any one of the preceding embodiments, wherein the construct does not induce antibody dependent cellular cytotoxicity in a cell comprising an Fc receptor.10. The construct of any one of the preceding embodiments, wherein the construct does not comprise an Fc domain.11. The construct of any one of the preceding embodiments, wherein the construct does not have antibody dependent cellular cytotoxicity (ADCC) activity.Atty. Dkt. No. 650053.0123812. The construct of any one of the preceding embodiments, wherein the construct has a sequence with at least 85% identity to one of SEQ ID NO: 7-9.13. The construct of any one of the preceding embodiments, wherein the construct comprises a linker between the anti-CD138 domain and the anti-CD47 domain.14. The construct of embodiment 13, wherein the linker comprises SEQ ID NO: 14.15. A polynucleotide comprising a sequence encoding the construct of any one of the preceding embodiments.16. The polynucleotide of embodiment 15, wherein the polynucleotide further comprises a chimeric antigen receptor (CAR) sequence.17. A cell comprising the polynucleotide of embodiment 15 or 16.18. The cell of embodiment 17, wherein the cell is a human cell.19. The cell of embodiment 18, wherein the cell is a human embryonic kidney (HEK) cell.20. The cell of embodiment 18. wherein the human cell is a chimeric antigen receptor (CAR) T cell.21. A pharmaceutical composition comprising the bispecific single-chain polypeptide construct of any one of embodiments 1-14, optionally, further comprising a suitable excipient.22. A pharmaceutical composition comprising the cell of embodiment 20.23. A method comprising contacting the construct of any one of embodiments 1-14 to a cell.24. The method of embodiment 23, wherein the cell is a human cell.25. A method comprising administering the pharmaceutical composition of embodiment 21 to a subject.26. A method of treating a disease or disorder associated with CD47 expression in a subj ect in need thereof, the method comprising administering a therapeutically effective amount of theAtty. Dkt. No. 650053.01238 pharmaceutical composition of embodiment 21 to the subject to treat the disease or disorder associated with CD47 expression in the subject.27. The method of embodiment 26, wherein the disease or disorder associated with CD47 expression is a cell proliferative disease or disorder.28. The method of embodiment 27, wherein the cell proliferative disease or disorder is multiple myeloma (MM).29. A method of treating a cell proliferative disease or disorder in a subj ect in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of embodiment 21 to the subject to treat the cell proliferative disease or disorder.30. The method of embodiment 29, wherein the cell proliferative disease or disorder is a cancer.31. The method of embodiment 30, wherein the cancer is multiple myeloma.32. The method of any one of embodiments 29-31, wherein the method further comprises administering a chemotherapy, a radiation therapy, a surgery, a hormone therapy, or an immunotherapy to the subject.33. The method of embodiment 32, wherein the immunotherapy comprises a chimeric antigen receptor (CAR) T cell therapy.34. The method of any one of embodiments 29-33. wherein the method increases the proportion of the construct binding to tumor cells as compared to an anti-CD47 antibody.35. The method of any one of embodiments 29-34, wherein the method reduces the proportion of the construct binding to T cells as compared to an anti-CD47 antibody.36. A method of treating a cell proliferative disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of embodiment 21 and a chimeric antigen receptor (CAR) T cell therapy to the subject to treat the cell proliferative disease or disorder in the subject.37. The method of embodiment 36, wherein the cell proliferative disease or disorder is a cancer.Atty. Dkt. No. 650053.0123838. The method of embodiment 37, wherein the cancer is multiple myeloma.39. The method of any one of embodiments 36-38, wherein the CAR T cell therapy is selected from the group consisting of axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, tisagenlecleucel, ciltacabtagene autoleucel, and idecabtagene vicleucel.40. The method of any one of embodiments 36-39, wherein the method further comprises administering a chemotherapy, a radiation therapy, a surgery7, a hormone therapy, or an immunotherapy to the subject.41. The method of any one of embodiments 36-40, wherein the method increases the proportion of the construct binding to tumor cells as compared to an anti-CD47 antibody.42. The method of any one of embodiments 36-41, wherein the method reduces the proportion of the construct binding to T cells as compared to an anti-CD47 antibody.43. The method of embodiment 42, wherein the T cells are CAR T cells.44. A method of treating a cell proliferative disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising cells expressing the construct of any one of embodiments 1-14.45. The method of embodiment 44, wherein the cells expressing the construct are T cells.46. The method of embodiment 45, wherein the T cells are CAR T cells.47. A method of making a CAR T cell that expresses a bispecific single chain construct, the method comprising contacting a T cell with the polynucleotide of embodiment 16.48. A method of making a CAR T cell that expresses a bispecific single chain construct, the method comprising contacting a CAR T cell with the polynucleotide of embodiment 15.49. A method of making a CAR T cell that expresses a bispecific single chain construct, the method comprising contacting a T cell with the polynucleotide of embodiment 15 and further contacting the T cell with a polynucleotide comprising a chimeric antigen receptor (CAR) sequence.EXAMPLESAtty. Dkt. No. 650053.01238

[0080] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.Example 1 - CD47-CD138 bispecific construct exhibits selective targeting of multiple myeloma

[0081] CD47 is a “do not eat me signal” that is overexpressed on malignant tumors including multiple myeloma (MM). CD47 on tumor cells interacts with SIRP-a on macrophages and inhibits its phagocytosis enabling tumor progression. Clinical studies with antibodies blocking CD47-SIRP-a interaction to promote tumor phagocytosis have shown varying success. CD47 is widely expressed on normal tissue and their binding to these monoclonal antibodies can lead to loss of treatment efficacy due to “sink effect” and to off-tumor on-target toxicity. In addition, when used as an adjuvant, CD47 antibody had a deleterious effect on CAR T cell survival and efficacy (Y amada-Hunter et al., Nature 2024).

[0082] To overcome these limitations and improve the on-tumor effect of CD47 blockade in MM, the inventors generated a bispecific construct wherein an anti-CD47 single chain variable fragment (scFv) is combined with an anti-CD138 scFv. Since CD138 expression is mostly limited to MM the inventors hypothesized that CD47-CD138 bispecific antibody (BsAb) construct will preferentially bind to MM cells, thereby improving anti-tumor efficacy and at the same time minimizing off-tumor toxicity.

[0083] After initially testing different combinations of linkers, the inventors generated CD47- CD138 BsAb construct (with and without Fc domain) using (Fig. 1) human serum albumin (HSA, AA 427-451) as a linker. As these antibodies have been reformatted into a bispecific construct, the inventors determined whether the bispecific construct was able to independently bind their cognate antigens. As expected, bispecific construct showed strong binding to either immobilized recombinant CD47 or CD138, similar to the corresponding monospecific scFv in solid phase time resolved fluorescence assay.

[0084] The inventors determined whether the bispecific construct can bind simultaneously to both CD47 and CD138 using a bridging ELISA assay. Recombinant CD138 was immobilized on a plate and incubated with either CD47 scFv or CD47-CD138 BsAb construct. After washing, the plate was incubated with biotinylated CD47, and binding was determined using streptavidin-HRP. Compared to PBS control (OD 0.048) or CD47 scFv (OD 0.078), CD47- CD138 BsAb construct (OD 2.95) was able to bind both antigens simultaneously (Fig 2).

[0085] The inventors determined whether CD47-CD138 BsAb construct had increased binding to MM cells compared to CD47 scFv (Fig. 3). OPM2 myeloma cell line was incubatedAtty. Dkt. No. 650053.01238 with CD47 scFv or CD47-CD138 BsAb construct, and binding was determined by flow cytometry. CD47-CD138 BsAb construct had a significantly increased binding to 0PM2 cells compared to CD47 scFv (median fluorescence intensity (MFI) 7.82E+05 vs 7.07E+04, p<0.0001, n=3). The inventors then determined whether this increased binding to MM cells will lead to increased phagocytosis. Monocytes isolated from peripheral blood were cultured in media with human serum for 7 days and then co-cultured with carboxyfluorescein succinimidyl ester (CFSE) labelled 0PM2 cells for 4 hours in the presence of equimolar concentrations of CD47 scFv or CD47-CD138 BsAb construct and phagocytosis was determined by flow cytometry' (Fig. 4). There was increased phagocytosis of 0PM2 cells in the presence of CD47-CD138 BsAb construct compared to CD47 scFv (12.2% vs 9.5%, p<0.05, n=2).

[0086] Next, the inventors compared the binding of CD47 scFv or CD47-CD138 BsAb construct to T cells and, as expected, the MFI values were similar (3.82E+04 vs 3.54E+04, n=3) with no significant difference. The inventors then determined whether CD47-CD138 BsAb construct would preferentially bind to MM cells in the presence of T cells. The inventors mixed an equal number of T cells and OPM2 cells and incubated them with CD47 scFv or CD47-CD138 BsAb construct and determined antibody binding by flow cytometry (Fig. 5). There was significantly increased binding of CD47-CD138 BsAb to 0PM2 cells compared to T cells (MFI 6.25E+05 vs 4.82E+04, p<0.0001, n=3). The inventors then determined whether CD47-CD138 BsAb construct would preferentially induce phagocytosis of MM cells over T cells. T cells labelled with Cell Trace Far Red (CTFR) dye and OPM2 cells labelled with CFSE were co-cultured with macrophages in the presence of CD47-CD138 BsAb construct (Fig. 6). Macrophages demonstrated preferential phagocytosis of OPM2 cells compared to T cells in the presence of CD47-CD138 BsAb construct (6. 1% vs 1.9%, p<0.03, n=2).

[0087] The inventors next determined whether CD47-CD138 BsAb would improve tumor control in a xenograft model of multiple myeloma. Luciferase expressing OPM2 cells (5xlOA4 cells / mice) were engrafted subcutaneously in NSG mice and after confirming engraftment by IVIS. Mice were treated with either PBS or CD47 scFv or CD47-CD138 BsAb intraperitoneally daily for 4 weeks. There was significantly less tumor growth in the CD47- CD138 BsAb treated mice compared to PBS or CD47 scFv treatment (Fig. 7).

[0088] The inventors have developed a CD47-CD138 BsAb that has enhanced and preferential binding to MM cells. The inventors have demonstrated that this preferential binding translates into augmented phagocytosis of MM cells while relatively sparing CD47 expressing T cells. Sparing normal cells expressing CD47 will possibly reduce off-tumorAtty. Dkt. No. 650053.01238 toxicities associated with CD47 targeted therapies, therefore, enhancing their efficacy. The inventors will now generate CAR T cells that can secrete CD47-CD138 BsAb construct and determine efficacy in myeloma in vitro and in vivo.

[0089] Methods

[0090] Antibodies transient expression and purification

[0091] The inventors generated monospecific antibodies with a single chain variable fragment (scFv) and bispecific antibodies (BsAb) in small and large scale. For small scale production the inventors transiently transfected LX293 cells using LipoFectamine reagent according to the manufacturer’s instructions (Thermo Fisher Scientific). After 72 h, the cell culture supernatants containing the antibodies were collected and concentrated using Amicon Centrifugal Filters (SigmaAldrich). For high yield protein production, the constructs were transiently expressed in Expi293 mammalian expression system according to the manufacturer’s instructions (Thermo Fisher Scientific).

[0092] The inventors generated human anti-CD47 scFv (Clone 5F9) and human anti-CD47- CD138 BsAb construct with or without the Fc receptor domain. Four different human anti- CD47-CD138 BsAb constructs were used to generate bispecific constructs with different linker sequences (LinkK, (648)3, CBH-1 and HSA). After 5-6 days, clarified cell cultures supernatants containing the antibodies were purified by affinity7chromatography using the Ni- NTA resin (Thermo Fisher Scientific) which allows high specificity and affinity purification of proteins carrying a His tag. After washing and elution steps, the purified proteins were dialyzed using Pur-A-Lyzer Maxi Dialysis tubes with a cutoff of 25kD (SigmaAldrich). Subsequently, the proteins were resuspended in IX DPBS 10% glycerol and sterile filtered. The protein concentration was determined by the Pierce BCA Protein Assay Kit according to the manufacturer’s instructions (Thermo Fisher Scientific). Protease Inhibitor Cocktail (SigmaAldrich) was added at 1:500 dilution in each step of the purification process. Correct protein size and purity was confirmed by Coomassie blue stain in SDS-PAGE gel.

[0093] Affinity measurements by bridging ELISA

[0094] The inventors determined whether the Bispecific construct can bind simultaneously to both CD47 and CD138 using a bridging ELISA assay. A transparent 96-well plate (Greiner- Bio One) was coated with recombinant human CD138 His Tag (AcroBiosystems) at 2.5ug / ml in IX PBS and incubated overnight. After washing and blocking steps 4ug / ml of biotinylated human CD47 (AcroBiosystems) was added and incubate 1 h at room temperature. After washing steps O. lug / ml streptavidin-HRP (AcroBiosystems) was added and incubate 1 h at room temperature. After following washes, TMB (Bio-Rad Laboratories) was added andAtty. Dkt. No. 650053.01238 incubated 5 min at room temperature. Finally, the reaction was stopped by adding stopping reaction solution. The plate was read on the Spark plate reader at 450nm (Tecan).

[0095] Flow cytometry to determine antibody binding

[0096] The inventors determined whether CD47-CD138 BsAb construct had increased binding to MM cells compared to CD47 scFv. OPM2 -plain and OPM2-mCherry-luc cell lines were incubated 20 min at room temperature with 1.3 uM of purified CD47 scFv or CD47- CD138 BsAb construct. Cells were washed once with FACS buffer and then stained 20 min on ice with anti-Flag (APC, 15, Biolegend, 1 :200) antibody. After staining, cells were washed twice with FACS buffer and analyzed on Cytek Aurora cytometer (Cytek Biosciences). Data were analyzed using FLOWJO software (BD).

[0097] The inventors then determined whether CD47-CD138 BsAb construct would preferentially bind to MM cells in the presence of T cells. The inventors mixed equal number of purified human T cells (Human T cells w ere isolated from PBMCs using MojoSort Human CD3 T Cell Isolation Kit) and OPM2 cells and incubated them 20 min at room temperature with 1.3 uM of purified CD47 scFv or CD47-CD138 BsAb construct. Cells were washed once with FACS buffer and then stained 20 min on ice with anti-Flag (APC, 15, Biolegend, 1 :200) antibody. After staining, cells were washed twice with FACS buffer and analyzed on Cytek Aurora cytometer (Cytek Biosciences). Data w ere analyzed using FLOWJO software (BD).

[0098] OPM2 phagocytosis assay

[0099] Monocytes were isolated from human PBMCs using EasySep Human Monocytes Isolation kit (StemCell) and differentiated into macrophages by 7 days of culture in IMDM media (Life Technologies) + 10% AB human serum (SigmaAldrich). All in-vitro phagocytosis assays were performed by co-culture target cells and macrophages at a ratio of 50,000 target cells and 25,000 macrophages in serum-free IMDM media for 4 h in a humidified, 5% CO2 incubator at 37°C in 96-well U-bottom suspension plates (Greiner-Bio One). Macrophages were harvested from plates using TrypLE Express solution (Life Technologies). After coculture, cells w ere incubated 5 min at room temperature with lOug / ml human Fc receptor blocking solution (BD) followed by 10 min incubation on ice to stop phagocytosis. Then, cells were washed once with FACS buffer and stained 20 min on ice with human anti-CDl lb (APC, MI / 70, Biolegend, 1 :200). After staining, cells were washed twice with FACS buffer and stained with viability dye DAPI (Biolegend) prior analysis on LSRFortessaX20 cytometer (BD). Data w ere analyzed using FLOWJO software (BD).

[0100] On the day of the experiment, target OPM2 cells were stained with CFSE dye (Invitrogen) by resuspending cells in PBS (1 mM working solution) for 20 min at 37°CAtty. Dkt. No. 650053.01238 protected from light and washed with FBS-containing media to absorb any unbound dye. OPM2-CFSE cells were then resuspended in serum-free IMDM media and incubated 20 min in a humidified, 5% CO2 incubator at 37°C with equimolar concentrations of purified CD47 scFv (1.3 uM) or CD47-CD138 BsAb construct (1.3 uM) or with PBS (control). Then, OPM2- CFSE treated cells were mixed with harvested human macrophages and incubated for 4 h as described above. Phagocytosis was determined as the number of CDl lb+, CFSE+ macrophages quantified as a percentage of the total CD1 lb+ macrophages by flow cytometry.

[0101] Preferential phagocytosis assay

[0102] The inventors then determined whether CD47-CD138 BsAb construct would preferentially induce phagocytosis of MM cells over T cells. Human T cells were isolated as described above from the same PBMCs donor from which monocytes were isolated to prevent cross reactivity and labelled with CTFR dye (Invitrogen) by resuspending cells in PBS (1 mM working solution) for 20 min at 37°C protected from light and washed with FBS-containing media to absorb any unbound dye. 0PM2 cells were labelled with CFSE dye as described above. Then, equal number of T-cells-CTFR and OPM2-CFSE labeled cells were combined and resuspended in serum-free IMDM media and incubated 20 min in a humidified, 5% CO2 incubator at 37°C with equimolar concentrations of purified CD47 scFv (1.3 uM) or CD47- CD138 BsAb construct (1.3 uM) or with PBS (control). Treated T-cells-CTFR and 0PM2- CFSE cells were mixed with harvested human macrophages and incubated for 4 h as described above.

[0103] After co-culture, cells w ere incubated 5 min at room temperature with 1 Oug / ml human Fc receptor blocking solution (BD) followed by 10 min incubation on ice to stop phagocytosis. Then, cells were washed once with FACS buffer and stained 20 min on ice with an antibody cocktail of human anti-CDl lb (PE, MI / 70, Biolegend, 1 :200) and human anti-CD3 (BV785, SK7, BD, 1 : 100). After staining, cells were washed twice with FACS buffer and stained with viability dye DAPI (Biolegend) prior analysis on LSRFortessaX20 cytometer (BD). Data w ere analyzed using FLOWJO software (BD). OPM2 phagocytosis was determined as the number of CD3-, CD1 lb+, CFSE+ macrophages quantified as a percentage of the total CD3-, CD1 lb+ macrophages, whereas T-cells phagocytosis was determined as the number of CD3-, CD1 lb+, CTFR+ macrophages quantified as a percentage of the total CD3-, CDl lb+ macrophages.

[0104] In vivo experiments

[0105] For OPM2 xenograft experiments, female 6-7-week-old immune-deficient NSG (NOD.Cg-PrkdcscldI12rgtml1A 11 / SzJ) mice were engrafted with 5xl04OPM2-mCherry-luc cells by subcutaneous injection of single cell suspension in 50% Matrigel Basement MembraneAtty. Dkt. No. 650053.01238Matrix (Coming) into the left flank of the mice. On day 3 after tumor cell injection, tumor implantation was confirmed using IVIS Spectrum instrument (PerkinElmer). Mice received an intraperitoneal injection of 3.3 mg D-luciferin (GoldBio), and bioluminescent images were acquired starting 10 min after the D-luciferin injection. Average radiance (p / s / cm2 / sr) was quantified using Living Image software version 4.8.2 (Revvity).

[0106] Mice were then randomized into treatment groups, receiving a daily dose via I.P. of 250ug produced in-house human anti-CD47 scFv, human anti-CD47-CD138 BsAb construct or PBS (control) for 4 weeks. Tumor grow th was monitored every week on the IVIS Spectrum instrument (PerkinElmer) as described above. Animals were daily monitored for signs of distress in accordance with institutional regulations and weekly w eighed.

[0107] No signs of toxicity were observed on either the CD47 scFv orthe CD47-CD138 BsAb construct treated mice groups and animal weights remain normal throughout the 4 weeks treatment.Example 2 - Generation and characterization of CD47-CD138 BsAb

[0108] Methods

[0109] Antibodies production and characterization

[0110] Antibodies transient expression and purification

[0111] For high yield protein production, the constructs were transiently expressed in Expi293 mammalian expression system according to the manufacturer's instructions (Thermo Fisher Scientific). Four different human anti-CD47-CD138 BsAb constructs were used to generate BsAbs with different linker sequences (LinkK, (G4S)s, CBH-1 and HSA (SEQ ID NO: 14)) (Fig. 8A). The inventors also generated human anti-CD47 scFv, human anti-CD47- CD138 BsAb with or without the Fc receptor (FIG. 8B) and human anti-CD138 scFv (FIG. 8C). After 5-6 days, clarified cell cultures supernatants containing the antibodies were purified by affinity chromatography using the Ni-NTA resin (Thermo Fisher Scientific) which allows high specificity and affinity purification of proteins carrying a His-tag. After washing and elution steps, the purified proteins were dialyzed using Pur-A-Lyzer Maxi Dialysis tubes with a cutoff of 25kD (SigmaAldrich). Subsequently, the proteins were resuspended in IX DPBS 10% glycerol and sterile filtered. The protein concentration was determined by the Pierce BCA Protein Assay Kit according to the manufacturer’s instructions (Thermo Fisher Scientific). Protease Inhibitor Cocktail (SigmaAldrich) w as added at 1:500 dilution in each step of the purification process. Correct protein size and purity was confirmed by Coomassie blue stain in SDS-PAGE gel.Atty. Dkt. No. 650053.01238

[0112] The expression of the BsAb was best when human serum albumin (HSA, AA 403- 427) was used as a linker compared to others like LinkK, (G4S)s or CBH-1 (8mg / ml. 2mg / ml, 3mg / ml and 6mg / ml, respectively).

[0113] Solid phase time resolved fluorescence (TRF) binding assay

[0114] As these antibodies have been reformatted into a BsAb, the inventors determined whether the BsAb was able to independently bind to its cognate antigens by TRF assay. Recombinant human CD47 (5ug / ml) and CD 138 (5ug / ml) were immobilized on black 96-well plate (Greiner Bio-One). Binding of antibodies was detected using anti-FLAG M2 (Sigma Aldrich) followed by incubation with an anti-mouse IgG-Europium antibody (Perkin Elmer). After incubation with DELFIA Enhancement solution (Perkin Elmer). TRF signal was determined on Tecan Spark plate reader

[0115] As expected, BsAb showed strong binding to either immobilized human recombinant CD47 or CD138, similar to the corresponding CD47 scFv (FIG. 9).

[0116] Affinity measurements by bridging ELISA

[0117] The inventors determined whether the BsAb can bind simultaneously to both CD47 and CD138 using a bridging ELISA assay. A transparent 96-well plate (Greiner-Bio One) was coated with recombinant human CD138 His Tag (AcroBiosystems) at 2.5ug / ml in IX PBS and incubated overnight. After washing and blocking steps 4ug / ml of biotinylated human CD47 (AcroBiosystems) was added and incubate 1 h at room temperature. After washing steps O. lug / ml streptavidin-HRP (AcroBiosystems) was added and incubate 1 h at room temperature. After following washes, TMB (Bio-Rad Laboratories) was added and incubated 5 min at room temperature. Finally, the reaction was stopped by adding stopping reaction solution. The plate was read in Tecan plate reader at 450nm (Biosystems). Compared to PBS control (OD 0.048) or CD47 scFv (OD 0.078), CD47-CD138 BsAb (OD 2.95) was able to bind both antigens simultaneously (FIG. 10).

[0118] Assessing CD47-CD138 BsAb quality

[0119] To determine CD47-CD138 BsAb preserves binding overtime, the inventors performed a cell-based binding assay by flow cytometry. The inventors incubated OPM2 cells with 1.3 uM of different batches of CD47-CD138 BsAb that were produced, purified and storage at different time points from the assay (1 month, 2 months, 3 months and 1 year). After 20min incubation the inventors washed the cells and stained with anti-FLAG-tag antibody (Biolegend). The results showed same level of CD47-CD138 BsAb binding regardless the time the BsAb was storage (median fluorescence intensity (MFI) 1 month: 1.25E6, MFI 2 months: 1.29E6, MFI 3 months: 1.32E6 and MFI 1 year: 1.20E6) (FIG. 11).Atty. Dkt. No. 650053.01238

[0120] Flow cytometry analysis

[0121] Cell lines

[0122] All cells were maintained in culture with RPMI-1640 medium (ATCC) containing 20% fetal bovine serum (FBS) (Thermo Fisher) and lOug / ml of penicillin-streptomycin (Gibco). Multiple myeloma (MM) cell lines OPM2 and MM1S were stained to verify the surface expression of CD138, CD47 and BCMA antigens. Cell lines were washed with FACS buffer (2% BSA in PBS) before the staining was performed in FACS buffer for 20 min on ice with commercially full-length antibodies human anti-CD138 (APC, MI15, BD, 1 :200), human anti-CD47 (APC, CC2C6, Biolegend, 1 :200) and anti-BCMA (APC, 19F2, Biolegend, 1 :200). After staining, cells were washed twice with FACS buffer and analyzed on Cytek Aurora cytometer (Cytek Biosciences). Data were analyzed using FLOWJO software (BD) (FIG. 12).

[0123] Cell-based binding assay with in-house produced antibodies

[0124] The inventors determined whether CD47-CD138 BsAb had increased binding to MM cells compared to CD47 scFv or CD138 scFv. OPM2 and MM1S cell lines were incubated 20 min at room temperature with 1.3 uM of purified CD47 scFv, CD138 scFv or CD47-CD138 BsAb. Cells were washed once with FACS buffer and then stained 20 min on ice with antiFlag (APC, 15, Biolegend, 1 :200) antibody. After staining, cells were washed twice with FACS buffer and analyzed on Cytek Aurora cytometer (Cytek Biosciences). Data were analyzed using FLOWJO software (BD). CD47-CD138 BsAb had increased binding to OPM2 cells compared to CD138 scFv and CD47 scFv (MFI 9.42E+05, 3.77E+05, 5.72E+04). The inventors observed similar results with MM I S since CD47-CD138 BsAb showed and increased binding to this cell line when compared to CD47 scFv or CD138 scFv (MFI 1.16E+05, 9.25E+04, 7.51E+04) (FIG. 13A). The inventors’ results showed that CD47-CD138 BsAb significantly binds to OPM2 compared to CD47 scFv (MFI 7.81E+05 vs 7.07E+04, p>0.001, n=3) (FIG. 13B).

[0125] Cell-based preferential binding assay

[0126] First, the inventors determined the binding of CD47 scFv or CD47-CD138 BsAb to T cells. Human T cells were isolated and incubated 20 min at room temperature with 1.3 uM of purified CD47 scFv or CD47-CD138 BsAb. Cells were washed once with FACS buffer and then stained 20 min on ice with anti-Flag (APC, 15, Biolegend, 1:200) antibody. After staining, cells were washed twice with FACS buffer and analyzed on Cytek Aurora cytometer (Cytek Biosciences). Data were analyzed using FLOWJO software (BD). As expected, there was no significant difference in binding between CD47 scFv and CD47-CD138 BsAb to T cells (MFI 2.77E+04 vs 3.81E+04, n=3) (FIG. 14).Atty. Dkt. No. 650053.01238

[0127] Next, the inventors determined whether CD47-CD138 BsAb would preferentially bind to MM cells in the presence of T cells. The inventors mixed equal number of purified human T cells and U266 cells and incubated them 20 min at room temperature with 0.01 uM (subsaturating concentration) of CD47 scFv or CD47-CD138 BsAb. Cells were washed once with FACS buffer and then stained 20 min on ice with a cocktail of anti-CD3 (BUV395, SK7, BD, 1 :200) and anti-Flag (APC, 15. Biolegend, 1 :200) antibodies. After staining, cells were washed twice with FACS buffer and analyzed on Cytek Aurora cytometer (Cytek Biosciences). Data were analyzed using FLOWJO software (BD). There was significantly increased binding of CD47-CD138 BsAb to U266 cells compared to T cells (MFI 1.57E+04 vs 9.21E+03, p<0.001, n=2) (FIG. 15).

[0128] Phagocytosis Assay

[0129] For all phagocytosis assays, this is the general protocol that is followed. Specific changes pertaining to the labelling of cells are described with each assay below.

[0130] Monocytes were isolated from human PBMCs using EasySep Human Monocytes Isolation kit (StemCell) and differentiated into macrophages by 7 days of culture in IMDM media (Life Technologies) + 10% AB human serum (SigmaAldrich). All in-vitro phagocytosis assays were performed by co-culture target cells and macrophages at a ratio of 50,000 target cells and 25,000 macrophages in serum-free IMDM media for 4 h in a humidified, 5% CO2 incubator at 37°C in 96-well U-bottom suspension plates (Greiner-Bio One). Macrophages were harvested from plates using TrypLE Express solution (Life Technologies). After coculture, cells were incubated 5 min at room temperature with lOug / ml human Fc receptor blocking solution (BD) followed by 10 min incubation on ice to stop phagocytosis. Then, cells were washed once with FACS buffer and stained 20 min on ice with human anti-CDl lb (APC, MI / 70, Biolegend, 1 :200). After staining, cells were washed twice with FACS buffer and stained with viability dye DAPI (Biolegend) prior analysis on LSRFortessaX20 cytometer (BD). Data were analyzed using FLOWJO software (BD).

[0131] On the day of the experiment, target OPM2 cells or isolated T-cells were stained with CFSE dye (Invitrogen) by resuspending cells in PBS (1 pM working solution) for 20 mm at 37°C protected from light and washed with FBS -containing media to absorb any unbound dye. OPM2-CFSE+ or T-cells-CFSE+ were then resuspended in serum-free IMDM media and incubated 20 min in a humidified, 5% CO2 incubator at 37°C with equimolar concentrations (1.3 uM) of purified CD47 scFv or CD47-CD138 BsAb or w ith PBS (control). Then, OPM2- CFSE+ or T-cells-CFSE+ treated cells were mixed with harv ested human macrophages andAtty. Dkt. No. 650053.01238 incubated for 4 h as described above. Phagocytosis was determined as the number of CD1 lb+, CFSE+ macrophages quantified as a percentage of the total CDl lb+ macrophages. Phagocytosis was normalized to the value obtained in the PBS group. There was increased phagocytosis of 0PM2 cells in the presence of CD47-CD138 BsAb compared to CD47 scFv and PBS control (mean of 0.978, 0.822 and 0.581, p<0.01 and p<0.006, n=4) (FIG. 16A) and T-cells phagocytosis showed no difference across groups (mean of 0.542, 0.558 and 0.612, n=4) (FIG. 16B).

[0132] Preferential phagocytosis assay

[0133] The inventors then determined whether CD47-CD138 BsAb would preferentially induce phagocytosis of MM cells over T cells. Human T cells were isolated as described above from the same PBMCs donor from which monocytes were isolated to prevent cross reactivity and labelled with CTFR dye (Invitrogen) by resuspending cells in PBS (1 pM working solution) for 20 min at 37°C protected from light and washed with FBS-containing media to absorb any unbound dye. 0PM2 cells were labelled with CFSE dye as described above. Then, equal number of T-cells-CTFR and OPM2-CFSE labeled cells were combined and resuspended in serum-free IMDM media and incubated 20 min in a humidified, 5% CO2 incubator at 37°C with equimolar concentrations (1.3 uM) of purified CD47scFv, CD47- CD138 BsAb or PBS control. Treated T-cells-CTFR and OPM2-CFSE cells were mixed with harvested human macrophages and incubated for 4 h as described above.

[0134] After co-culture, cells were incubated 5 min at room temperature with lOug / ml human Fc receptor blocking solution (BD) followed by 10 min incubation on ice to stop phagocytosis. Then, cells were washed once with FACS buffer and stained 20 min on ice with an antibody cocktail of human anti-CDl lb (PE, MI / 70, Biolegend, 1 :200) and human anti-CD3 (BV785, SK7, BD, 1 : 100). After staining, cells were washed twice with FACS buffer and stained with viability dye DAPI (Biolegend) prior analysis on LSRFortessaX20 cytometer (BD). Data were analyzed using FLOWJO software (BD). OPM2 phagocytosis was determined as the percentage of CD3-, CDl lb+, CFSE+ macrophages quantified as a percentage of the total CD3-, CD1 lb+ macrophages, whereas T-cells phagocytosis was determined as the number of CD3-, CDl lb+, CTFR+ macrophages quantified as a percentage of the total CD3-, CDl lb+ macrophages. Phagocytosis was normalized to the value obtained in the PBS group. Macrophages demonstrated preferential phagocytosis of OPM2 cells as results showed enhance phagocytosis of 0PM2 cells in the presence of CD47-CD138 BsAb compared toAtty. Dkt. No. 650053.01238CD47scFv or PBS control (mean of 0.968, 0.829 and 0.648, p<0.001, p<0.01, n=4) while T- cells were spared (mean of 0.552, 0.560 and 0.550, n=4) (FIG. 17A and 17B).

[0135] Phagocytosis of myeloma cells lacking BCMA antigen

[0136] OPM2-BCMAKOcells generation

[0137] Antigen escape is a major problem in many cancer immunotherapies. Multiple myeloma patients can relapse even after BCMA-CAR-T cells treatment due to low. mutated or absence of BCMA expression in the surface of myeloma cells. To mimic this environment in-vitro, the inventors first generated OPM2-BCMAKOand U266-BCMAKOcells by knocking out BCMA using CRISPR-CAS9.

[0138] BCMA negative (or knock out, BCMAK0) population was sorted using MACSQuant Tyto sorter (Miltenyi). At day 7 after sorting, the inventors confirmed absence of BCMA expression in the sorted OPM2-BCMAKOand U266-BCMAKOcells compared to the cells in the untransduced condition by staining with human anti-BCMA antibody as previously described (FIG. 18).

[0139] Phagocytosis assay of MM cells that escape anti -BCMA-CAR-T cells cytotoxicity

[0140] Then, the inventors performed phagocytosis assay as previously described by mixing equal numbers of OPM2V'1. OPM2-BCMAKOstained with CTFR dye, anti-BCMA-CAR-T cells and macrophages in the presence of equimolar concentrations (1.3uM) of CD47-CD138 BsAb, CD47 scFv or PBS. OPM2-BCMAKOphagocytosis was determined as the percentage of CDl lb+, CTFR+ cells. Macrophages demonstrated relatively enhanced phagocytosis of OPM2-BCMAKOin the presence of CD47-CD138 BsAb compared to CD47 scFv and significantly enhanced phagocytosis when compared to PBS control (mean of 12.2, 11.1 and 7.3, CD47-CD138 BsAb vs PBS p<0.001, n=3) (FIG. 19). These results demonstrated that CD47-CD138 BsAb enhanced tumor phagocytosis even when MM cells escape anti-BCMA- CAR-T cell cytotoxicity due to lack of BCMA expression.

[0141] Generation of anti-BCMA-CAR-T cells secreting CD47-CD138 BsAb or CD47 scFv

[0142] Lentiviral vectors (BsAb secretion)

[0143] The inventors generated plasmids that contains CD47-HSA-CD138 or CD47 scFv sequence combined with the HER2t extracellular transmembrane domain or the control plasmid containing only the HER2t sequence (SFG-CD47-HSA-CD138-HER2t)1. The inventors have named these plasmids pMWHer2t CD47-HSA-CD138 scFv, pMWHer2t CD47 scFv and pMWHer2t control.Atty. Dkt. No. 650053.01238

[0144] '[Johnson, Adam J et al. “Rationally Designed Transgene-Encoded Cell-Surface Polypeptide Tag for Multiplexed Programming of CAR T-cell Synthetic Outputs / ’ Cancer immunology research vol. 9,9 (2021), which is incorporated by reference herein],

[0145] Retroviral vector (BCMA CAR)

[0146] The CAR sequence of the SFG-BCMA-CAR retroviral plasmid encodes the MMLV promoter followed by the BCMA scFv. hemagglutinin tag, CD8a hinge and transmembrane domains, the 4- IBB costimulatory domain, and the CD3C, signaling domain. In addition, there is GFP separated from the CAR by the P2A ribosomal skip sequence.

[0147] CAR T cell manufacturing by lenti and retroviral double transduction

[0148] Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by density-gradient centrifugation using Ficoll-Paque Plus (Fisher). Next, T-cells were isolated from 10xl0A6 PBMCs as previously described and resuspended in AIM V media (ThermoFisher) containing 5% human A / B serum (Sigma- Aldrich), lOug / ml penicillin-streptomycin and 400IU / ml IL-2 (R&D Systems). Next, T-cells were prepared at a concentration of 250,000 T-cells / ml and prewashed CD3 / CD28 activator beads (Thermo-Fisher) were added at a ratio of 1: 1. T-cells were plated in a 24-well suspension plate and incubated for 48h before transduction. One day prior double transduction, RETRONECTIN plate was prepared by coating 24-well suspension plate with 500ul of 20ug / ml RETRONECTIN reagent (Takara) working solution diluted with IX DPBS and incubating it at 4C overnight.

[0149] On the day of transduction, 500ul of concentrated retroviral supernatant was added to RETRONECTIN coated 24-well suspension plate and centrifugated at 32C for 2h at 2,000g. AIM V media was carefully replaced on the T-cells plate with fresh media containing 6 pg / ml polybrene together with 50ul of concentrated lentiviral supernatants. After the RETRONECTIN coated plate centrifugation finished, retroviral supernatant was discarded, and previously prepared T-cells were transferred to the RETRONECTIN coated plate to perform double transduction via spinoculation at 21C for Ih at 1000g. T-cells were resuspended and then returned to the incubator for another 24h. One day after the double transduction, 1 ml of complete AIM-V media was replaced, IL-2 was renewed, and cells were diluted to a concentration of 0.4xl0A6 cells / ml.

[0150] At day 3 post transduction, the inventors determined transduction efficiency by flow cytometry. Retroviral transduction efficiency was determined by GFP expression whereas lentiviral transduction efficiency was determined by staining cells with human anti- ErbB2 / Her2 (AF647, Hu5, R&D. 1 :25) antibody. Therefore, double transduced anti- BCMAGFP+CAR-T cells expressing HER2 (marker for the gene expression encoded in theAtty. Dkt. No. 650053.01238 lentiviral constructs) were identified as GFP+, HER2+ and sorted as previously described. Before sorting. CD3 / CD28 activating beads were magnetically removed and replaced after sorting at a ratio of 3: 1 to allow cell expansion.

[0151] Double-transduced CAR-T cells were expanded keeping cell concentration at 0.4 *10A6 cells / ml and adding complete media and IL-2 accordingly. At day 8-12 of expansion, CD3 / CD28 activating beads were removed from the culture and cells were frozen in 90% fetal calf serum containing 10% dimethyl sulfoxide or used immediately for downstream assays. For some experiments, T cells underwent the production process without addition retroviral and lentiviral particles.

[0152] Characterization of anti-BCMA-CAR-T cells secreting CD47-CD138 BsAb

[0153] Western blot assay

[0154] The three generated CAR-T cells previously described (anti-BCMA-CAR-T cells secreting CD47-CD138 BsAb, anti-BCMA-CAR-T cells secreting CD47scFv and anti- BCMA-CAR-T cells), were revived and expanded for 10 days as previously described. Next, CD3 / CD28 activating beads were removed, cells were pellet down and saved for downstream experiments. Cell culture supernatants were collected to identify the corresponding secreted proteins (CD47-CD138 BsAb and CD47scFv) by western blot analysis. At day 10 of expansion, the number of cells and volume of supernatants collected from each CAR-T cell type were approximately 15xlOA6 cells in 30ml of culture media.

[0155] From each cell culture supernatant, the inventors purified and concentrated the secreted proteins (CD47-CD138 BsAb and CD47scFv) using Ni-NTA resin as previously described. The inventors performed the same protocol using anti-BCMA-CAR-T cell culture supernatant as a negative control and as internal control by splitting anti-BCMA-CAR-T cell culture into two fractions and spiking one of them with CD47-CD138 BsAb and CD47scFv that were produced and purified using the Expi293 mammalian expression system. The internal control helped to determine the expected size and whether the purification protocol implemented captured and purified the secreted scFv from CAR-T cells supernatants (FIG. 20A).

[0156] After protein purification, protein denaturation was performed by NuPAGE LDS following manufacturer’s instructions (Thermo Fisher) and samples were run in a 4-20% 1.5mm Bis-Tris Nu-PAGE gel (Invitrogen). Next, the inventors performed a lOmin semi-dry protein transfer using the Power Blotter Transfer Stack instrument (Invitrogen). Then, the inventors blocked the membrane for Ih at room temperature with IX Pierce fast blocking buffer (Thermo Fisher) and incubated overnight at 4C with primary antibody anti-mouseAtty. Dkt. No. 650053.01238FLAG M2 (1 :1,000 dilution) (Thermo Fisher). The next day, the inventors washed the membrane before incubation with secondary antibody anti -mouse IRDye680RD (1: 10,000 dilution) (Licor) for Ih at room temperature. Finally, the inventors washed and dried the membrane for visualization on a gel imager instrument (Azure Biosystems 600). Results confirmed that the protocol used for protein purification from cell culture supernatants is reliable as the inventors obtained the expected size bands in the internal controls. Importantly, the inventors identified the bands corresponding to the secreted CD47-CD138 BsAb and CD47scFv from CAR-T cell culture supernatants. Moreover, as expected, no bands were observed in the negative control sample (FIG. 20B).

[0157] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0158] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.SequencesAtty. Dkt. No. 650053.01238

Claims

1. Atty. Dkt. No. 650053.01238CLAIMS1. A bispecific single-chain polypeptide construct comprising an anti-CD47 domain and an anti-CD138 domain.

2. The construct of claim 1, wherein the anti-CD47 domain comprises a first heavy chain and a first light chain and the anti-CD138 domain comprises a second heavy chain and a second light chain.

3. The construct of claim 1 or 2, wherein the anti-CD47 domain comprises SEQ ID NO: 1.

4. The construct of claim 1. wherein the anti-CD138 domain comprises SEQ ID NO: 4.

5. The construct of claim 1, wherein the first heavy chain comprises SEQ ID NO: 2.

6. The construct of claim 1. wherein the first light chain comprises SEQ ID NO: 3.

7. The construct of claim 1, wherein the second heavy chain comprises SEQ ID NO: 5.

8. The construct of claim 1. wherein the second light chain comprises SEQ ID NO: 6.

9. The construct of claim 1, wherein the construct does not induce antibody dependent cellular cytotoxicity in a cell comprising an Fc receptor.

10. The construct of claim 1, wherein the construct does not comprise an Fc domain.

11. The construct of claim 1, wherein the construct does not have antibody dependent cellular cytotoxicity (ADCC) activity.

12. The construct of claim 1, wherein the construct has a sequence with at least 85% identity to one of SEQ ID NOs: 7-9.

13. The construct of claim 1, wherein the construct comprises a linker between the anti- CD138 domain and the anti-CD47 domain.

14. The construct of claim 13, wherein the linker comprises SEQ ID NO: 14.

15. A polynucleotide comprising a sequence encoding the construct of claim 1.Atty. Dkt. No. 650053.0123816. The polynucleotide of claim 15, wherein the polynucleotide further comprises a chimeric antigen receptor (CAR) sequence.

17. A cell comprising the polynucleotide of claim 15 or 16.

18. The cell of claim 17, wherein the cell is a human cell.

19. The cell of claim 18, wherein the cell is a human embry onic kidney (HEK) cell.

20. The cell of claim 18. wherein the human cell is a chimeric antigen receptor (CAR) T cell.

21. A pharmaceutical composition comprising the bispecific single-chain polypeptide construct of claim 1, optionally, further comprising a suitable excipient.

22. A pharmaceutical composition comprising the cell of claim 20.

23. A method comprising contacting the construct of claim 1 to a cell.

24. The method of claim 23, wherein the cell is a human cell.

25. A method comprising administering the pharmaceutical composition of claim 21 to a subject.

26. A method of treating a disease or disorder associated with CD47 expression in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 21 to the subject to treat the disease or disorder associated with CD47 expression in the subject.

27. The method of claim 26. wherein the disease or disorder associated with CD47 expression is a cell proliferative disease or disorder.

28. The method of claim 27, wherein the cell proliferative disease or disorder is multiple myeloma (MM).

29. A method of treating a cell proliferative disease or disorder in a subj ect in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 21 to the subject to treat the cell proliferative disease or disorder.Atty. Dkt. No. 650053.0123830. The method of claim 29, wherein the cell proliferative disease or disorder is a cancer.

31. The method of claim 30, wherein the cancer is multiple myeloma.

32. The method of claim 29. wherein the method further comprises administering a chemotherapy, a radiation therapy, a surgery, a hormone therapy, or an immunotherapy to the subject.

33. The method of claim 32, wherein the immunotherapy comprises a chimeric antigen receptor (CAR) T cell therapy.

34. The method of claim 29, wherein the method increases the proportion of the construct binding to tumor cells as compared to an anti-CD47 antibody.

35. The method of claim 29, wherein the method reduces the proportion of the construct binding to T cells as compared to an anti-CD47 antibody.

36. A method of treating a cell proliferative disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 21 and a chimeric antigen receptor (CAR) T cell therapy to the subject to treat the cell proliferative disease or disorder in the subject.

37. The method of claim 36, wherein the cell proliferative disease or disorder is a cancer.

38. The method of claim 37, wherein the cancer is multiple myeloma.

39. The method of claim 36, wherein the CAR T cell therapy is selected from the group consisting of axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, tisagenlecleucel, ciltacabtagene autoleucel, and idecabtagene vicleucel.

40. The method of claim 36, wherein the method further comprises administering a chemotherapy, a radiation therapy, a surgery, a hormone therapy, or an immunotherapy to the subject.

41. The method of claim 36, wherein the method increases the proportion of the construct binding to tumor cells as compared to an anti-CD47 antibody.

42. The method of claim 36, wherein the method reduces the proportion of the construct binding to T cells as compared to an anti-CD47 antibody.Atty. Dkt. No. 650053.0123843. The method of claim 42, wherein the T cells are CAR T cells.

44. A method of treating a cell proliferative disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising cells expressing the construct of claim 1.

45. The method of claim 44, wherein the cells expressing the construct are T cells.

46. The method of claim 45, wherein the T cells are CAR T cells.

47. A method of making a CAR T cell that expresses a bispecific single chain construct, the method comprising contacting a T cell with the polynucleotide of claim 16.

48. A method of making a CAR T cell that expresses a bispecific single chain construct, the method comprising contacting a CAR T cell with the polynucleotide of claim 15.

49. A method of making a CAR T cell that expresses a bispecific single chain construct, the method comprising contacting a T cell with the polynucleotide of claim 15 and further contacting the T cell with a polynucleotide comprising a chimeric antigen receptor (CAR) sequence.

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