Chimeric antigen receptors to control HIV infection
Inventors
Berger, Edward A. • Ghanem, Mustafa H. • Dey, Barna
Assignees
US Department of Health and Human Services
Publication Number
US-10246505-B2
Publication Date
2019-04-02
Expiration Date
2034-11-25
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Abstract
The present disclosure is directed to novel multispecific chimeric antigen receptor (CAR) proteins and DNA sequences encoding these proteins. The CARs comprise at least two extracellular domains fused, via a transmembrane domain to a cytoplasmic signaling domain comprising two signaling domains. The disclosure further relates to nucleic acids encoding the novel CARs, to host cells expressing the novel CARs, and to methods of using the CARs to co-stimulate effector functions in the cells and for using cells expressing the receptors for treatment of disease and viral infections. The disclosure also relates to methods of generating a recombinant T cell with reduced susceptibility to HIV infection.
Core Innovation
This disclosure provides novel multispecific chimeric antigen receptor (CAR) proteins comprising at least two extracellular targeting domains fused via a transmembrane domain to a cytoplasmic signaling domain that includes two signaling domains. The CAR proteins include multispecific targeting segments that can bind to HIV Env or fragments thereof, such as gp120. These CARs can be used to generate host cells, such as T cells, expressing the CARs for the co-stimulation of effector functions and treatment of disease and viral infections, particularly human immunodeficiency virus (HIV) infection.
The problem being solved arises from limitations in current HIV treatments. While antiretroviral therapy has improved the quality of life and durably suppresses HIV-1 replication, strict adherence to treatment is required and replication-competent virus persists in long-lived reservoirs, making antiretroviral therapy alone non-curative. The disclosure addresses the need for alternative therapies that enable cessation of antiretroviral therapy by providing genetically engineered immune cells expressing multispecific CARs that selectively target and kill HIV-infected cells, while reducing susceptibility of the effector cells to HIV infection.
The multispecific CAR proteins described include an N-terminal extracellular targeting segment with a first targeting domain derived from CD4, binding to HIV Env, and a second targeting domain comprising either a carbohydrate recognition domain (CRD) derived from a human C-type lectin or a single-chain antibody variable fragment (scFv) that binds different sites on HIV Env. The linkage between targeting domains can include short linkers that prevent simultaneous binding to a single gp120 molecule, enhancing activity through serial triggering. The disclosure also relates to nucleic acids encoding such CAR proteins and methods of producing recombinant T cells with reduced HIV susceptibility.
Claims Coverage
The claims disclose 17 inventive features primarily centered on multispecific chimeric antigen receptor proteins, their structural components, and methods of use related to HIV treatment.
Multispecific chimeric antigen receptor extracellular targeting segment
The CAR protein comprises an N-terminal extracellular targeting segment with two distinct binding domains: a CD4 domain comprising the D1 and D2 extracellular domains of CD4, and a carbohydrate recognition domain (CRD) from a human C-type lectin (MBL2 or Langerin) that both bind different sites on HIV Env and together confer binding to HIV Env.
Linker connecting targeting domains
The CAR protein includes a linker connecting the first targeting domain (CD4) to the second targeting domain (CRD), with the linker length optionally no more than 20 amino acids and in some embodiments ten amino acids long.
Specific amino acid sequences for targeting domains
The CAR includes extracellular domain sequences selected from SEQ ID NO: 51 (Langerin CRD) or SEQ ID NO: 53 (MBL2 CRD).
Complete CAR protein structure
The CAR protein further includes a linker connecting the extracellular targeting segment to a transmembrane domain, the transmembrane domain itself, and an intracellular region containing both a cytoplasmic co-stimulatory signaling domain and a cytoplasmic effector function signaling domain, all functionally linked.
Use of CD28 and CD3 zeta domains in CAR components
The transmembrane domain originates from CD28, the cytoplasmic co-stimulatory signaling domain is from CD28, and the cytoplasmic effector function signaling domain is from CD3 zeta.
CAR protein with MBL2 targeting domain and associated intracellular domains
A CAR protein comprising the N-terminal extracellular targeting segment with a CD4 domain and an MBL2 domain, a CD28 transmembrane region, and intracellular co-stimulatory and effector signaling domains from CD28 and CD3 zeta, respectively.
T cell expressing the multispecific CAR protein
A T cell, including CD8+ and/or CD4+ T cells, engineered to express the multispecific CAR protein as defined in claim 1.
Reduced susceptibility of T cells to HIV infection
The T cells engineered to express the multispecific CAR protein are characterized by reduced susceptibility to HIV infection.
Composition comprising CAR-expressing T cells
A pharmaceutical composition comprising the CAR-expressing T cells and a carrier suitable for therapeutic administration.
Method of killing HIV-infected cells
A method comprising contacting the CAR-expressing T cell composition with HIV-infected cells expressing gp120, resulting in targeted killing of the HIV-infected cells.
Method of reducing HIV-infected cells in a subject
Administering a therapeutically effective amount of the CAR-expressing T cell composition to a subject infected with HIV to reduce the level of HIV-infected cells.
The claims comprehensively cover multispecific CAR proteins featuring CD4 and lectin-based carbohydrate recognition domains targeting distinct sites on HIV Env, with specified linkers and intracellular signaling domains. The claims extend to T cells expressing these CARs, their pharmaceutical compositions, and methods for killing HIV-infected cells and treating HIV-infected subjects with reduced risk of HIV infection of the effector T cells.
Stated Advantages
Greater potency due to two independent targeting moieties recognizing distinct conserved sites on HIV Env.
Reduced susceptibility of CAR-expressing T cells to HIV infection, preventing effector cells from becoming HIV reservoirs.
Potential for CARs to provide persistent targeted killing of HIV-infected cells, including cells activated from latent reservoirs.
Minimized immunogenicity by incorporation of human-derived components, reducing the likelihood of anti-idiotypic antibody responses.
Documented Applications
Use of multispecific CAR-expressing T cells for binding and killing HIV-infected cells expressing gp120.
Treatment of HIV-infected subjects by administration of recombinant T cells expressing multispecific CARs to reduce viral burden.
Generation of CAR-expressing T cells with reduced susceptibility to HIV infection, improving safety and therapeutic efficacy.
Prevention of HIV infection mother-to-infant transmission and post-exposure prophylaxis using CAR-expressing cells.
Combination therapies including CAR-expressing cells with antiretroviral agents and immunomodulators for treatment of HIV disease.
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