US-12617832-B2 - Genetic engineering of B cell receptors and uses thereof in antigen-induced antibody secretion

US12617832B2US 12617832 B2US12617832 B2US 12617832B2US-12617832-B2

Abstract

The present invention relates to methods and compositions for engineering B cells to express transgenic B cell receptor (BCR) for antigen-induced antibody secretion, compositions, methods and uses thereof in immunotherapy.

Inventors

Adi Barzel
Alessio D. NAHMAD
Tal AKRIV
Miriam Fried
Iris Dotan
Daniel NATAF

Assignees

RAMOT AT TEL-AVIV UNIVERSITY LTD.

Dates

Publication Date: 20260505
Application Date: 20210312

Claims (8)

1 . A method of genetically engineering a primary mammalian cell of the B lineage for expression of an engineered B cell receptor (BCR) and for antigen-induced secretion of an antibody of interest or antigen binding fragment thereof or chimera thereof from said cell and or progeny cell thereof, said method comprising a step of contacting a primary mammalian cell of the B lineage with at least one nucleic acid cassette comprising at least one nucleic acid sequence of interest, or with any vector or vehicle comprising said cassette, wherein said nucleic acid sequence of interest comprises at least one nucleic acid sequence coding 5′ to 3′ for (i) at least one variable domain of an immunoglobulin heavy chain or any binding fragment or chimera thereof, and (ii) at least one variable domain of an immunoglobulin light chain or any binding fragment or chimera thereof, and at least one splice donor site (SD), wherein said cassette is flanked on the 5′ and 3′ ends thereof by universal homology arms for integrating by homologous recombination, wherein said universal homology arms target the insertion of said nucleic acid cassette comprising said nucleic acid sequence of interest into a target genomic sequence within the immunoglobulin heavy chain (IgH) locus, downstream to the last segment of the J region of said IgH locus and upstream of an enhancer region of said IgH locus of said heavy chain of said BCR, and wherein said method genetically engineers the primary cell of the B cell lineage to express an engineered BCR and to secrete the antibody of interest or antigen-binding fragment thereof or chimera thereof by antigen-induced activation from said cell and or progeny thereof.
2 . The method according to claim 1 , wherein said target genomic sequence within the IgH locus is located at least 100 nucleotides downstream to the J region of the variable domain.
3 . The method according to claim 1 , wherein said cassette further comprises at least one exogenous hotspot motif for somatic hypermutations, said somatic hypermutations retaining or minimally changing the protein translated from said nucleic acid sequence.
4 . The method according to claim 1 , wherein said cassette is flanked on at least one of the 5′ and 3′ ends thereof by at least recognition sites for a site-specific nuclease, wherein the insertion of said nucleic acid sequence of interest into the target genomic locus is mediated by said site-specific nuclease, and optionally, said nuclease is at least one programmable engineered nuclease (PEN), wherein said PEN comprises at least one clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR associated (cas) protein system, and wherein said method further comprises the step of contacting said cell with at least one of: (a) one or more of CRISPR/cas protein, or any nucleic acid molecule encoding said Cas protein; and (b) one or more of nucleic acid sequence comprising at least one guide RNA (gRNA) that targets the insertion of said nucleic acid sequence of interest into a target genomic sequence within the IgH locus, or any nucleic acid sequence encoding said gRNA; or any kit, composition or vehicle comprising at least one of (a) and (b).
5 . The method according to claim 1 , wherein said contacting the cell with said at least one nucleic acid cassette is performed in a mammalian subject, the method comprising a step of administering to said subject an effective amount of the at least one nucleic acid cassette comprising said at least one nucleic acid sequence of interest or of any vector or vehicle comprising said cassette.
6 . The method according to claim 5 , wherein said mammalian subject is suffering from a pathologic disorder.
7 . The method according to claim 1 , wherein said antibody of interest or antigen binding fragment or chimera thereof comprises at least one antibody of interest or antigen binding fragment or chimera thereof that is directed against an antigen associated with a pathologic disorder, said antigen comprising a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen, or a tumor associated antigen (TAA).
8 . A genetically engineered mammalian cell of the B lineage engineered to express an engineered B cell receptor (BCR) and to secrete an antibody of interest or an antigen-binding fragment thereof upon antigen induction, or a population of said engineered cells, or any composition thereof, said cell comprising at least one exogenous nucleic acid sequence of interest coding for at least one variable domain of an immunoglobulin light chain and at least one variable domain of an immunoglobulin heavy chain of said antibody of interest or an antigen-binding fragment thereof upstream of an at least one splice donor site (SD), integrated within the IgH locus downstream to the last segment of the J region of the variable domain and upstream of an enhancer region of said IgH locus of said heavy chain, optionally, wherein said nucleic acid sequence coding for the at least one variable domain of at least one of an immunoglobulin light chain and an immunoglobulin heavy chain, further comprises at least one exogenous hotspot motif for somatic hypermutation, wherein said somatic hypermutation retains or minimally changes the protein translated from said nucleic acid sequence.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application is a continuation-in-part of international application no. PCT/IL2019/051026, filed Sep. 12, 2019, insofar as it designates the United States, which in turn claims benefit of U.S. provisional applications 62/730,561, filed Sep. 13, 2018, and 62/840,429, filed Apr. 30, 2019. The entire contents of each of these applications are hereby incorporated herein by reference. FIELD OF THE INVENTION The invention relates to gene therapy, specifically, immunotherapy. More specifically, the invention relates to methods and compositions for engineering B cells to express transgenic B cell receptor (BCR) for antigen-induced antibody secretion, compositions, methods and uses thereof in immunotherapy. SEQUENCE LISTING INCORPORATION BY REFERENCE The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 12, 2021, is named P2021-03-12SequenceListing_BARZEL2A_ST25.txt and is 220 kilobytes in size. BACKGROUND ART References considered to be relevant as background to the presently disclosed subject matter are listed below: [1] Luo X M, et al. Blood. 2009; 113(7):1422-1431.[2] Fusil F, et al. Mol Ther. 2015; 23(11):1734-1747.[3] US20160289637[4] Lin Y C, et al., EMBO J. 2018 Aug. 7. pii: e99243. doi: 10.15252/embj.201899243.[5] Greiner, V. et al. iScience 12, 369-378 (2019).[6] Voss, J. E. et al. Elife (2019). doi:10.7554/eLife.42995.[7] Hartweger, H. et al. Journal of Experimental Medicine 216, 1301-1310 (2019).[8] Moffett, H. F. et al. Sci. Immunol. 4, (2019).[9] WO 2019/028417.[10] Laskov, R., et al. Mol. Immunol. 48, 733-745 (2011)[11] Yeap, L. S. et al. Cell 163, 1124-1137 (2015).[12] Hwang, J. K. et al. Proc. Natl. Acad. Sci. 201709203 (2017). doi:10.1073/pnas.1709203114 Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter. BACKGROUND OF THE INVENTION Monoclonal Antibodies are used widely to treat diverse medical conditions, including cancer autoimmune diseases. However, monoclonal Antibody therapy is rarely curative. An Antibody has a limited half-life and has to be repeatedly administered at a very high financial cost and with aggravated risk for the development of anti-drug antibodies. The genetic engineering of B cells for antibody secretion may overcome these limitations, but previous attempts used mostly lentiviral vectors and transposons, which integrate promiscuously and do not allow natural activation of the transgenic B cell receptor (BCR) for antigen-induced antibody secretion. More specifically, B cells naturally express BCRs on their cell membranes. Upon specific binding of an appropriate antigen to the BCR, the B cell may be activated, and it may differentiate into an antibody secreting plasma cell. Antigen-induced activation is a tightly regulated process, which takes place mainly in germinal centers, and which requires the participation of dedicated T cells and dendritic cells. Importantly, antigen-induced B cell activation in the germinal centers allows for affinity maturation, class switch recombination and memory retention. Several methods have been published regarding cell engineering for antibody secretion. Luo et al. have engineered human hematopoietic stem/progenitor cells to produce an anti-HIV Ab upon in vitro maturation to B cells [1]. However, expression levels were constitutively low as the lentivector-coded Ab did not allow B cell activation. Fusil et al. [2] used a different lentivector design that allowed B cell activation and differentiation into Ab producing plasma cells. However, the promiscuous and ectopic lentiviral integration as well as the synthetic poly adenyaltion sites that were used prevented native regulation on the transition from BCR expression to antibody secretion and did not allow for affinity maturation upon activation. Goldberg et al., [3] used genome editing to engineer B cells at the IgH locus. However, this publication only discusses introducing an antibody rather than engineering the BCR and therefore antigen induced activation, affinity maturation, class switch recombination and memory retention were not demonstrated. Bastisda et al. [4] describe in vivo CRISPR/Cas9 nuclease mediated strategy to generate knock-in mice of a DNA fragment bearing a pre-arranged human B-cell receptor heavy chain into the native murine immunoglobulin locus. This manipulation is performed in fertilized oocytes thereby manipulating germline B cells that result in deletion of the D4 to J1-4 endogenous segments in all B cells produced in the transgenic mice. This publication however, does not concerns engineering of B cells, nor any use thereof in gene therapy, specifically, the use of the engineered B cells as a therapeutic tool in a mammalian subject. More recently, ef