# US 2019/0169289 A1 · Optimized pne-based chimeric receptor t cell switches and uses thereof

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- Number: US 2019/0169289 A1
- Document ID: US20190169289A1
- Status: Published application
- Kind: A1
- Application number: 15/566,680
- Filed: 2016-04-15
- Published: 2019-06-06
- Earliest claimed: 2015-04-15
- Assignees: The California Institute for Biomedical Research
- Applicants: The California Institute for Biomedical Research
- Inventors: Travis YOUNG; David T. RODGERS; Ian HARDY; Chanhyuk KIM; Peter G. SCHULTZ; Eric HAMPTON; Eduardo LABORDA; Leonard PRESTA
- Web page: https://patentgrep.com/patent/US20190169289A1

## CPC

- C07K 16/2803: {against the immunoglobulin superfamily}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4205: {Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4}
- A61K 40/421: {Immunoglobulin superfamily}
- A61K 40/4211: {CD19 or B4}
- A61K 40/4221: {CD20}
- A61K 40/4242: {Transcription factors, e.g. SOX or c-MYC}
- A61P 35/00: Antineoplastic agents
- C07K 14/39: from yeasts
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/18: against material from animals or humans
- C07K 16/2851: {against the lectin superfamily, e.g. CD23, CD72}
- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C07K 16/32: against translation products of oncogenes
- A61K 2039/505: {comprising antibodies}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- A61K 2039/585: {wherein the target is cancer}
- A61K 2039/80: {Vaccine for a specifically defined cancer}
- A61K 2239/23: On/off switch
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C07K 2317/53: Hinge
- C07K 2317/55: Fab or Fab'
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/00: Fusion polypeptide
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Disclosed herein are chimeric antigen receptor effector cells (CAR-ECs) and CAR-EC switches. The switchable CAR-ECs are generally T cells. The one or more chimeric antigen receptors may recognize a peptidic antigen on the CAR-EC switch. The CAR-ECs and switches may be used for the treatment of a condition in a subject in need thereof.

## Claims

### Claim 1

A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain an intracellular signaling domain, wherein the extracellular domain comprises:
a. a region that interacts with a chimeric antigen receptor switch; and
b. a hinge domain.

### Claim at position 2

-15. (canceled)

### Claim 16

A soluble T cell receptor switch comprising:
a. a chimeric antigen receptor binding peptide; and
b. a soluble T cell receptor or portion thereof.

### Claim at position 4

-25. (canceled)

### Claim 26

A chimeric antigen receptor switch comprising:
A.
i. a chimeric antigen receptor binding peptide;
ii. a targeting moiety;
iii. and a linker,
wherein the linker links the chimeric antigen receptor binding peptide to the targeting moiety;
B.
i. a fusion antibody comprising:
a. a chimeric antigen receptor binding peptide; and
b. a light chain of an antibody or antibody fragment; wherein the chimeric antigen receptor binding peptide is connected to the light chain of the targeting antibody or antibody fragment, and
ii. a heavy chain of the targeting antibody or antibody fragment; or
C.
i. a fusion antibody comprising:
a. a chimeric antigen receptor binding peptide; and
b. a heavy chain of an antibody or antibody fragment; wherein the chimeric antigen receptor binding peptide is connected to the heavy chain of the targeting antibody or antibody fragment, and
ii. a light chain of the targeting antibody or antibody fragment.

### Claim at position 6

-72. (canceled)

### Claim 73

A method of producing an optimal switchable chimeric antigen receptor platform, comprising:
A.
i. incorporating a first chimeric antigen receptor binding peptide to a first site of a targeting moiety that binds a first cell surface molecule on a first target cell to produce a first switch;
ii. incorporating a second chimeric antigen receptor binding peptide to a second site of a second targeting moiety that binds a second cell surface molecule on a second target cell to produce a second switch;
iii. contacting the first target cell with the first switch and a first chimeric antigen receptor effector cell expressing a first chimeric antigen receptor;
iv. contacting the second target cell with the second switch and a second chimeric antigen receptor effector cell expressing a second chimeric antigen receptor; and
v. comparing a first cytotoxic effect of the first switch and the first chimeric antigen receptor effector cell on the first target cell to a second cytotoxic effect of the second switch and the second chimeric antigen receptor effector cell on the second target cell; and
vi. selecting the first switch and first chimeric antigen receptor effector cell or the second switch and the second chimeric antigen receptor effector cell as the optimal switchable chimeric antigen receptor platform based on comparing the first cytotoxic effect to the second cytotoxic effect: or
B. modulating a parameter of a first switchable chimeric antigen receptor platform or a second swtichable chimeric antigen receptor platform, and comparing a first outcome of the first switchable chimeric antigen receptor platform in response to modulationg the parameter to a second outcome of the second switchable chimeric antigen receptor platform in response to modulationg the parameter, wherein the parameter is selected from:
i. affinity of the chimeric antigen receptor for the chimeric antigen receptor-effector cell switch;
ii. distance between the chimeric antigen receptor-effector cell membrane and a target cell;
iii. a number of chimeric antigen receptor-effector cell binding peptides on the chimeric antigen receptor-effector cell switch;
iv. chimeric antigen receptor density on the chimeric antigen receptor-effector cell membrane;
v. number of engagements between the chimeric antigen receptor-effector cell and the target cell,
vi. and any combination thereof.

### Claim at position 8

-106. (canceled)

### Claim 107 (depends on 73)

An optimized chimeric antigen receptor-effector cell platform, comprising:
a. a chimeric antigen receptor-effector cell switch comprising a chimeric antigen receptor binding peptide and a targeting moiety; and
b. a chimeric antigen receptor-effector cell that expresses a chimeric antigen receptor, wherein the chimeric antigen receptor-effector cell platform is derived by the method of claim 73.

### Claim at position 10

-129. (canceled)

### Claim 130 (depends on 73)

A method of treating a condition in a subject in need thereof, comprising administering to the subject the chimeric antigen receptor switch and the chimeric antigen receptor effector cell of an optimized chimeric antigen receptor-effector cell platform, wherein the optimized chimeric antigen receptor-effector cell platform, comprises:
a. a chimeric antigen receptro-effector cell switch comprising a chimeric antigen receptor binding peptide and a targeting moiety; and
b. a chimeric anatigen receptor-effector cell that expresses a chimeric antigen receptor, wherein the chimeric antigen receptor-effector cell platform is derived by the method of claim 73.

### Claim at position 12

-146. (canceled)

### Claim 147

A method of treating relapsed cancer, comprising administering:
a. a first chimeric antigen receptor switch comprising a first chimeric antigen receptor binding peptide and a first targeting moiety;
b. a second chimeric antigen receptor switch comprising a second chimeric antigen receptor binding peptide and a second targeting moiety; and
c. a chimeric antigen receptor effector cell that binds the first chimeric antigen receptor binding peptide and the second CAR-BP,
wherein the first chimeric antigen receptor switch is administered before a relapse of the subject and the second chimeric antigen receptor switch is administered after the relaspe of the subject.

### Claim at position 14

-149. (canceled)

### Claim 150

A two-component cancer therapeutic comprising:
a. chimeric antigen receptor switch comprising a chimeric antigen receptor binding peptide and a targeting moiety, wherein the chimeric antigen receptor binding peptide is a non-endogenous peptide; and
b. a chimeric antigen receptor effector cell that binds the chimeric antigen receptor binding peptide.

### Claim at position 16

-153. (canceled)

### Claim 154

A method comprising,
administering to a subject an optimized first chimeric antigen receptor effector cell (CAR-EC) platform comprising
a. a first CAR-EC switch comprising
i. a first chimeric antigen receptor binding peptide (CAR-BP) and
ii. a targeting moiety that binds a cell surface molecule on a target cell; and
b. a first CAR-EC comprising a first chimeric antigen receptor (CAR) that binds the CAR-BP on the CAR-EC switch;
wherein one or more parameter of the first CAR and the first CAR-EC switch are modified as compared to a second CAR and a second CAR-EC switch to induce an optimal CAR-EC-mediated cytotoxic effect on the target cell upon the binding of both the targeting moiety of the first CAR-EC switch to the surface molecule on the target cell and the first CAR-BP of the first CAR-EC switch to the first CAR on the first CAR-EC.

### Claim 155 (depends on 154)

The method of claim 154, wherein the parameter is selected from:
a. affinity of the CAR for the CAR-EC switch;
b. distance between the CAR-EC membrane and a target cell;
c. a number of chimeric antigen receptor-effector cell binding peptides (CAR-BPs) on the CAR-EC;
d. CAR density on the CAR-EC membrane;
e. number of engagements between the CAR-EC and the target cell; and
f. combinations of one or more of parameters a-e.

### Claim 156 (depends on 155)

The method of claim 155, wherein the distance between the CAR-EC membrane and a target cell is between 100 Å and 200 Å.

### Claim 157 (depends on 155)

The method of claim 155, wherein the CAR-EC comprises a short hinge that is about 10 to about 20 amino acids and a CAR-BP comprising a length of between about 10 and about 20 amino acids.

### Claim 158 (depends on 154)

The method of claim 154, wherein the spatial orientation of the immunological synapse is also optimized by modifying the grafting position of the first CAR-BP on the first CAR-EC switch.

### Claim 159 (depends on 158)

The method of claim 158, wherein the spatial orientation of the immunological synapse is optimized by comparing the cytotoxic effect of the first CAR-EC platform to one or more second CAR-EC platform, each second CAR-EC platform comprising a second CAR-EC switch that comprises a different spatial orientation of CAR-BP than the orientation on the first CAR-EC switch, and wherein each second CAR-BP on each second CAR-EC switch is the same CAR-BP that is on the first CAR-EC switch.

### Claim 160 (depends on 154)

The method of claim 154, wherein the first CAR-EC switch comprises more than one CAR-BP and wherein the number of such CAR-BPs on the first CAR-EC switch is optimized; wherein the number of CAR-BPs on the first CAR-EC switch is optionally optimized by comparing the cytotoxic effect of the first CAR-EC platform to one or more second CAR-EC platform, each second CAR-EC platform comprising a CAR-EC switch that comprises a different number of CAR-BPs than the number on the first CAR-EC switch, and wherein each CAR-BP on each second CAR-EC switch is the same CAR-BP that is on the first CAR-EC switch.

### Claim 161 (depends on 154)

The method of claim 154, wherein the CAR-EC is a modified T cell.

### Claim 162 (depends on 154)

The method of claim 154, wherein the CAR-BP comprises a peptide selected from a non-human peptide, a non-mammalian peptide, a synthetic non-naturally occurring peptide, a non-endogenous peptide, a non-immunogenic peptide, and a peptide that has low immunogenicity in a human subject.

### Claim 163 (depends on 154)

The method of claim 154, wherein the targeting moiety is selected from a protein, a peptide, an antibody, an antibody fragment, a small molecule, and a soluble T cell receptor or portion thereof.

### Claim 164 (depends on 154)

The method of claim 154, wherein the targeting moiety comprises an antibody or antigen-binding fragment thereof that binds a cell surface molecule selected from CD19, CD20, CD22, CD33, CEA, CLL1, BCMA, CS1, CD123 Her2, or a sequence selected from SEQ ID NOS: 21-38, or a portion thereof, or a homolog thereof.

### Claim 165 (depends on 154)

The method of claim 154, wherein the targeting moiety comprises a CD19 antibody or antigen-binding fragment thereof and wherein the CAR-BP is attached to the N-terminus of the heavy chain, the light chain, or both the heavy and the light chains.

### Claim 166 (depends on 165)

The method of claim 165, wherein the CAR-BP comprises a GCN4 peptide that is at least 85% identical to SEQ ID NO: 3.

### Claim 167 (depends on 165)

The method of claim 165, wherein the CAR-BP is attached to a portion of the targeting moiety via a linker peptide.

### Claim 168 (depends on 165)

The method of claim 165, wherein the CAR comprises an anti-GCN4 antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is optionally an anti-GCN4 scFv, or wherein the CAR comprises a short hinge, a short-dimeric hinge, or comprises a hinge selected from SEQ ID NOS: 70-71.

### Claim 169 (depends on 154)

The method claim 154, wherein the number of CAR-BPs is between about 1 and about 8.

### Claim 170 (depends on 154)

The method of claim 154, wherein the CAR density is a magnitude of about 100 to about 10̂10 CAR per CAR-EC.

### Claim 171 (depends on 154)

The method of claim 154, wherein the number of engagements is about 1 to about 100.

### Claim 172 (depends on 154)

The method of claim 154, further comprising administering to the subject one or more second CAR-EC switch, each second CAR-EC switch comprising:
a. a CAR-BP; and
b. a second targeting moiety that binds a cell surface molecule on a target cell;
wherein the second targeting moiety of each second CAR-EC switch differs from the first targeting moiety comprised on the first CAR-EC switch; and
wherein the CAR-BP comprised on the second CAR-EC switch is optionally
(i) the same as the first CAR-BP comprised on the first CAR-EC switch or
(ii) a second CAR-BP that differs from the first CAR-BP comprised on the first CAR-EC switch;
provided that if the second CAR-EC switch comprises a second CAR-BP, the CAR-EC platform further comprises a second CAR-EC comprising a CAR that binds to the second CAR-BP of the second CAR-EC switch.

### Claim 173

A method comprising, administering to a subject
a) a first CAR-EC switch comprising
i. a first chimeric antigen receptor binding peptide (CAR-BP) and
ii. a targeting moiety that binds a cell surface molecule on a target cell; and
b) a first CAR-EC comprising a first chimeric antigen receptor (CAR) that binds the CAR-BP on the CAR-EC switch;
wherein one or more parameter of the first CAR and the first CAR-EC switch are modified as compared to a second CAR and a second CAR-EC switch to induce an optimal CAR-EC-mediated cytotoxic effect on the target cell upon the binding of both the targeting moiety of the first CAR-EC switch to the surface molecule on the target cell and the first CAR-BP of the first CAR-EC switch to the first CAR on the first CAR-EC.