# US 10,246,505 B2 · Chimeric antigen receptors to control HIV infection

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- Number: US 10,246,505 B2
- Document ID: US10246505B2
- Status: Granted
- Kind: B2
- Application number: 15/039,384
- Filed: 2014-11-25
- Published: 2017-09-21
- Granted: 2019-04-02
- Earliest claimed: 2013-11-25
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Inventors: Edward A. Berger; Mustafa H. Ghanem; Barna Dey
- Web page: https://patentgrep.com/patent/US10246505B2

## CPC

- C07K 14/70514: {CD4}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70521: {CD28, CD152}
- C07K 14/70564: {Selectins, e.g. CD62}
- C07K 16/1145: {Env proteins, e.g. gp41, gp110/120, gp160, V3, principal neutralising domain [PND] or CD4-binding site}
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## 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.

## Claims

### Claim 1

A multispecific chimeric antigen receptor protein, comprising:
an N-terminal extracellular targeting segment comprising:
a first targeting domain comprising a CD4 domain that binds to HIV Env, wherein the CD4 domain comprises the D1 and D2 extracellular domains of CD4 comprising amino acids 1-208 of SEQ ID NO: 53; and
a second targeting domain comprising a carbohydrate recognition domain (CRD) from a human C-type lectin that binds to HIV Env, wherein the CRD is selected from the group consisting of a CRD from mannose-binding lectin 2 (MBL2) comprising amino acids 214-330 of SEQ ID NO: 53 and a CRD from Langerin comprising amino acids 214-354 of SEQ ID NO: 51;
wherein the first and second targeting domains bind to different sites on HIV Env, and wherein the multispecific chimeric antigen receptor protein binds to HIV Env.

### Claim 2 (depends on 1)

The multispecific chimeric antigen receptor protein of claim 1, comprising a linker connecting the first targeting domain to the second targeting domain.

### Claim 3 (depends on 1)

The multispecific chimeric antigen receptor protein of claim 1, comprising an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 51, and the amino acid sequence of SEQ ID NO: 53.

### Claim 4 (depends on 1)

The multispecific chimeric antigen receptor protein of claim 1, further comprising:
a linker connecting the extracellular targeting segment to a transmembrane domain;
the transmembrane domain; and
an intracellular region, comprising:
a cytoplasmic co-stimulatory signaling domain; and
a cytoplasmic effector function signaling domain,
wherein the transmembrane domain is linked or fused to the intracellular region.

### Claim 5 (depends on 4)

The multispecific chimeric antigen receptor protein of claim 4, wherein:
the transmembrane domain is from CD28;
the cytoplasmic co-stimulatory signaling domain is from CD28; and/or
the cytoplasmic effector function signaling domain is from CD3 zeta.

### Claim 6 (depends on 2)

The multispecific chimeric antigen receptor protein of claim 2, wherein the linker connecting the first targeting domain to the second targeting domain is no more than 20 amino acids long.

### Claim 7 (depends on 6)

The multispecific chimeric antigen receptor protein of claim 6, wherein the linker connecting the first targeting domain to the second targeting domain is ten amino acids long.

### Claim 8 (depends on 1)

The multispecific chimeric antigen receptor protein of claim 1, comprising:
the N-terminal extracellular targeting segment comprising:
the first targeting domain; and
the second targeting domain, wherein the second targeting domain is a MBL2 domain;
a transmembrane region from CD28; and
an intracellular region, comprising:
a cytoplasmic co-stimulatory signaling domain from CD28; and
a cytoplasmic effector function signaling domain from CD3 zeta,
wherein the N-terminal extracellular targeting segment is linked to the transmembrane region and the transmembrane region is linked or fused to the intracellular region.

### Claim 9 (depends on 8)

The multispecific chimeric antigen receptor protein of claim 8, wherein:
the N-terminal extracellular targeting segment comprises the amino acid sequence of SEQ ID NO: 53;
the transmembrane region from CD28 comprises the amino acid sequence of SEQ ID NO: 55;
the cytoplasmic co-stimulatory signaling domain from CD28 comprises the amino acid sequence of SEQ ID NO: 58; and
the cytoplasmic effector function signaling domain from CD3 zeta comprises the amino acid sequence of SEQ ID NO: 56.

### Claim 10 (depends on 1)

A T cell comprising the multispecific chimeric antigen receptor protein of claim 1.

### Claim 11 (depends on 10)

The T cell of claim 10, wherein the T cell is a CD8+ T cell and/or a CD4+ T cell.

### Claim 12 (depends on 10)

The T cell of claim 10, wherein the T cell is not susceptible to HIV infection.

### Claim 13 (depends on 10)

A composition, comprising the T cell of claim 10 and a carrier.

### Claim 14 (depends on 13)

A method of killing HIV-infected cells, comprising:
contacting the composition of claim 13 with an HIV-infected cell expressing gp120, thereby killing the HIV-infected cells.

### Claim 15 (depends on 13)

A method of reducing the level of HIV infected cells in a subject infected with HIV, comprising:
administering to the subject a therapeutically effective amount of the composition of claim 13, thereby treating the subject infected with HIV.

### Claim 16 (depends on 15)

The method of claim 15, wherein the T cell in the composition is a T cell that is not susceptible to HIV infection.

### Claim 17 (depends on 13)

A method of treating a subject with an HIV infection, comprising: administering to the subject a therapeutically effective amount of the composition of claim 13 under conditions sufficient to form an immune complex of the extracellular targeting segment on the chimeric antigen receptor protein and the extracellular domain of an HIV Env protein in the subject.