US-12618076-B2 - Methods of engineering platelets for targeting circulating tumor cells

US12618076B2US 12618076 B2US12618076 B2US 12618076B2US-12618076-B2

Abstract

Disclosed herein are nucleic acid constructs that can be used to build genetic circuits for producing antibodies comprising split toxins. Also disclosed herein are methods of producing platelets comprising the antibodies. The platelets produced by the methods disclosed herein can be used to target circulating tumor cells.

Inventors

Tara Deans

Assignees

UNIVERSITY OF UTAH RESEARCH FOUNDATION

Dates

Publication Date: 20260505
Application Date: 20200930

Claims (12)

1 . A nucleic acid construct comprising: a) a promoter operatively linked to a sequence encoding c-MYC, BMI1, and BCL-XL; and b) a sequence encoding an engineered antibody sequence, wherein the engineered antibody sequence comprises a split toxin sequence flanked by an intein sequence; wherein the sequence encoding c-MYC, BMI1, and BCL-XL is flanked by a first recombination site and a second recombination site; wherein the sequence encoding the engineered antibody sequence is out of frame from the promoter; wherein, when exposed to a recombinase under conditions suitable to catalyze a site-specific recombination event between the first recombination site and the second recombination site, the sequence encoding c-MYC, BMI1, and BCL-XL is excised and the sequence encoding the engineered antibody sequence is brought in frame with the promoter.
2 . The nucleic acid construct of claim 1 , wherein the promoter is CMV, RSV, U6, beta actin, or elongation factor promoter.
3 . The nucleic acid construct of claim 1 , wherein the first or second recombination sites are loxP, attP or Bxb1 recombination sites.
4 . The nucleic acid construct of claim 1 , wherein the promoter is a regulatable promoter.
5 . The nucleic acid construct of claim 1 , wherein the engineered antibody sequence comprises a split toxin sequence flanked by an intein fragment, wherein the intein fragment comprises an intein N-fragment, wherein the split toxin sequence is N-terminal to an intein N-fragment.
6 . The nucleic acid construct of claim 1 , wherein the engineered antibody sequence comprises a split toxin sequence flanked by an intein fragment, wherein the intein fragment comprises an intein C-fragment, wherein the split toxin sequence is C-terminal to an intein C-fragment.
7 . A megakaryocyte comprising the nucleic acid construct claim 1 .
8 . A method of producing a platelet comprising an engineered antibody, the method comprising: a. providing pluripotent stem cells comprising the nucleic acid construct of claim 1 ; b. culturing the pluripotent stem cells in a media under conditions to permit the expansion of the pluripotent stem cells to megakaryocytes; c. exposing the megakaryocytes to a recombinase under conditions suitable to catalyze a site specific recombination event between the first recombination site and the second recombination site, thereby excising the sequence encoding c-MYC, BMI1, and BCL-XL, and bringing the sequence encoding the engineered antibody sequence in frame with the promoter, and d. differentiating the megakaryocytes into platelets; wherein the platelets comprise the engineered antibody encoded by the engineered antibody sequence.
9 . The method of claim 8 , wherein the engineered antibody comprises: a. an Fc region that comprises a split toxin sequence flanked by an intein fragment, wherein the intein fragment comprises an intein N-fragment, and wherein the split toxin sequence is N-terminal to an intein N-fragment; b. an Fc region comprising a split toxin sequence flanked by an intein fragment, wherein the intein fragment comprises an intein C-fragment, and wherein the split toxin sequence is C-terminal to an intein C-fragment; or c. a first Fc region comprising a first split toxin sequence flanked by a first intein fragment, wherein the first intein fragment comprises an intein N-fragment, wherein the first split toxin sequence is N-terminal to the intein N-fragment, and a second Fc region comprising a second split toxin sequence flanked by a second intein fragment, wherein the second intein fragment comprises an intein C-fragment, wherein the second split toxin sequence is C-terminal to the intein C-fragment.
10 . The method of claim 8 , wherein the promoter is a regulatable promoter.
11 . The method of claim 10 , wherein the media comprises isopropyl β-D-1-thiogalactopyranoside (IPTG).
12 . The method of claim 9 , wherein the Fc region or the first Fc region and the second Fc region are Fc regions of an IgG antibody or fragment thereof, and wherein the IgG antibody or fragment thereof is an anti-Her-2 antibody.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 371 of International Application No. PCT/US2020/053445, filed on Sep. 30, 2020, which claims the benefit of the filing date of U.S. Provisional Application No. 62/908,874, which was filed on Oct. 1, 2019. The content of this these earlier filed applications is hereby incorporated by reference herein in its entirety. INCORPORATION OF THE SEQUENCE LISTING The present application contains a sequence listing that was submitted in ASCII format via EFS-Web concurrent with the filing of the application, containing the file name 21101_0376U2_SL.txt, which is 470 bytes in size, created on Mar. 24, 2022, and is herein incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5). BACKGROUND Cancer is the second leading cause of death in the United States. Every year, more than a million people are diagnosed with cancer and more than half a million people succumb to the disease (Society A C. Atlanta: 2012). More than 90% of cancer-associated deaths are caused by metastasis (Lou X L, et al., Chin J Cancer Res 2015; 27(5): 450-60). Although cancer continues to be a highly active research area, curative treatments remain elusive. A fundamental challenge in treating patients diagnosed with cancer is preventing metastasis because once cancer spreads, it is difficult to control (Gay L J, Felding-Habermann B. Nat Rev Cancer 2011; 11(2): 123-34). Current treatments for metastatic cancer generally require systemic therapy, such as chemotherapy, with the goal of stopping or slowing the growth of cancer or to relieve symptoms caused by it (Gkountela S, et al., ESMO Open 2016; 1(4): e000078). Systemic treatments are harsh for many patients because they can cause other health problems including nausea, vomiting, neuropathy, organ and tissue damage, and immune deficiencies (Love R R, et al., Cancer 1989; 63(3): 604-12). Similarly, patients using targeted drugs invariably face relapse and develop drug resistance, mostly due to the activation of alternative pathways (Diaz L A, Jr., et al. Nature 2012; 486(7404): 537-40; and Misale S, et al. Nature 2012; 486(7404): 532-6). Additionally, due to the varying level of cancer specific biomarker expression between patients, drugs that are effective for some patients may be ineffective or have severe side effects for others (Lenz H J. Advances in experimental medicine and biology 2006; 587: 211-31). Therefore, therapies that can adapt to different patients and eradicate a wide range of tumor cells while avoiding systemic side effects are highly desirable. SUMMARY Disclosed herein are nucleic acid constructs comprising: a) a promoter operatively linked to: i) a first recombination site; ii) c-MYC, BMI1, and BCL-XL; and iii) a second recombination site; and b) a sequence capable of encoding an engineered antibody sequence, wherein the engineered antibody sequence comprises a split toxin sequence flanked by an intein sequence, and wherein the sequence capable of encoding the engineered antibody sequence is out of frame from the promoter. Disclosed herein are megakaryocytes comprising a nucleic acid construct, wherein the nucleic acid construct comprises a promoter operatively linked to: i) a first recombination site; ii) a second recombination site; and iii) a sequence capable of encoding an engineered antibody sequence, wherein the engineered antibody sequence comprises a split toxin sequence flanked by an intein sequence, wherein the sequence capable of encoding the engineered antibody sequence is in frame with the promoter. Disclosed herein are engineered megakaryocytes comprising: a) an engineered antibody comprising an Fc region comprising a split toxin sequence flanked by an intein fragment, wherein the intein fragment comprises an intein N-fragment, and wherein the split toxin sequence is N-terminal to an intein N-fragment; b) an engineered antibody comprising an Fc region comprising a split toxin sequence flanked by an intein fragment, wherein the intein fragment comprises an intein C-fragment, and wherein the split toxin sequence is C-terminal to an intein C-fragment; or c) an engineered antibody comprising a first Fc region comprising a first split toxin sequence flanked by a first intein fragment, wherein the first intein fragment comprises an intein N-fragment, wherein the first split toxin sequence is N-terminal to the intein N-fragment and a second Fc region comprising a second split toxin sequence flanked by a second intein fragment, wherein the second intein fragment comprises an intein C-fragment, wherein the second split toxin sequence is C-terminal to the intein C-fragment. Disclosed herein are engineered platelets comprising: a) an engineered antibody comprising an Fc region that comprises a split toxin sequence flanked by an intein fragment, wherein the intein fragment comprises an intein N-fragment, and wherein the split toxin sequence is N-terminal to a