# US 11,111,288 B2 · Conditionally active chimeric antigen receptors for modified t-cells

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- Number: US 11,111,288 B2
- Document ID: US11111288B2
- Status: Granted
- Kind: B2
- Application number: 16/125,302
- Filed: 2018-09-07
- Published: 2019-01-10
- Granted: 2021-09-07
- Earliest claimed: 2014-08-28
- Assignees: BioAtla, Inc.
- Applicants: BioAtla, LLC
- Inventors: Jay M. Short; Hwai Wen Chang; Gerhard Frey
- Web page: https://patentgrep.com/patent/US11111288B2

## CPC

- C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- 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 40/4202: {Receptors, cell surface antigens or cell surface determinants}
- A61K 40/4212: {CD22, BL-CAM, siglec-2 or sialic acid binding Ig-related lectin 2}
- A61P 35/00: Antineoplastic agents
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/70503: {Immunoglobulin superfamily}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70521: {CD28, CD152}
- C07K 14/70535: {Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)}
- C07K 16/18: against material from animals or humans
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C12N 5/0636: {T lymphocytes}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 2039/572: {cytotoxic response}
- C07K 2317/31: multispecific
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- 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
- C12N 2501/515: CD3, T-cell receptor complex
- C12N 2510/00: Genetically modified cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- 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)

## Abstract

This disclosure relates to a chimeric antigen receptor for binding with a tumor specific target antigen. The chimeric antigen receptor comprises at least one antigen specific targeting region evolved from a parent protein or a fragment thereof and having a decrease in activity in the assay at the normal physiological condition compared to the activity in the assay under the aberrant condition. A method for producing the chimeric antigen receptor is also provided.

## Claims

### Claim 1

A chimeric antigen receptor for binding with a tumor specific target antigen, comprising:
i. at least one antigen specific targeting region evolved from a parent or wild-type protein or a domain thereof and having a decrease in activity in an assay at a normal physiological condition compared to the activity of the antigen specific targeting region in an assay at an aberrant condition that deviates from the normal physiological condition;
ii. a transmembrane domain; and
iii. an intracellular signaling domain;
wherein the tumor specific target antigen is selected from the group consisting of tyrosine kinase growth factor receptors that are overexpressed on a surface of a cancer cell.

### Claim 2

A chimeric antigen receptor for binding with a tumor specific target antigen, comprising:
i. at least one antigen specific targeting region evolved from a parent or wild-type protein or a domain thereof and having a decrease in activity in an assay at a normal physiological condition compared to the activity of the antigen specific targeting region in an assay at an aberrant condition that deviates from the normal physiological condition;
ii. a transmembrane domain; and
iii. an intracellular signaling domain;
wherein the tumor specific target antigen is selected from Axl and ROR2 .

### Claim 3 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the at least one antigen specific targeting region is selected from an antibody, a fragment of an antibody, a single chain antibody, a divalent single chain antibody or a diabody, a ligand, a receptor binding domain of a ligand, a receptor, a ligand binding domain of a receptor, and an affibody.

### Claim 4 (depends on 2)

The chimeric antigen receptor of claim 2, wherein the tumor specific target antigen is Axl and the at least one antigen specific targeting region is a single chain antibody having an amino acid sequence selected from SEQ ID NOS: 9-12.

### Claim 5 (depends on 2)

The chimeric antigen receptor of claim 2, wherein the tumor specific target antigen is ROR2 and the at least one antigen specific targeting region is a single chain antibody having an amino acid sequence of SEQ ID NO: 15.

### Claim 6 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the normal physiological condition and aberrant condition are a same condition selected from temperature, pH, osmotic pressure, osmolality, oxidative stress, an electrolyte concentration, a concentration of a small organic molecule, a concentration of inorganic molecule, cell types, and nutrient availability.

### Claim 7 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the normal physiological condition is a normal physiological pH in blood plasma of a mammalian subject and the aberrant condition is a pH in a tumor microenvironment.

### Claim 8 (depends on 7)

The chimeric antigen receptor of claim 7, wherein the normal physiological pH is in a range of from greater than 7.0 to about 7.8.

### Claim 9 (depends on 8)

The chimeric antigen receptor of claim 8, wherein the normal physiological pH is in the range of from about 7.2 to about 7.6.

### Claim 10 (depends on 7)

The chimeric antigen receptor of claim 7, wherein the aberrant pH is in a range of from 6.0 to less than 7.0.

### Claim 11 (depends on 10)

The chimeric antigen receptor of claim 10, wherein the aberrant pH is in the range of from 6.0 to about 6.8.

### Claim 12 (depends on 1)

The chimeric antigen receptor of claim 1, further comprising an extracellular spacer domain or at least one co-stimulatory domain.

### Claim 13 (depends on 12)

The chimeric antigen receptor of claim 12, wherein the extracellular spacer domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence and combinations thereof.

### Claim 14 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the at least one antigen specific targeting region has a ratio of activity at the aberrant condition to the same activity at the normal physiological condition of at least about 2.

### Claim 15 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the at least one antigen specific targeting region comprises two antigen specific targeting regions that are connected with a linker.

### Claim 16 (depends on 15)

The chimeric antigen receptor of claim 15, wherein the two antigen specific targeting regions each bind with a different target antigen or a different epitope of the same target antigen.

### Claim 17 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and transmembrane domains of a Type I transmembrane protein, an alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

### Claim 18 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the intracellular signaling domain is selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of a Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

### Claim 19 (depends on 1)

The chimeric antigen receptor of claim 1, further comprising a co-stimulatory domain selected from the group consisting of co-stimulatory domains of proteins in the TNFR superfamily, CD28, CD137, CD134, DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, and B7-H3.

### Claim 20 (depends on 1)

An expression vector, comprising a polynucleotide sequence encoding the chimeric antigen receptor of claim 1.

### Claim 21 (depends on 20)

The expression vector of claim 20, wherein the expression vector is selected from the group consisting of lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno associated virus vectors, adenovirus vectors, pox virus vectors, herpes virus vectors, engineered hybrid viruses, and transposon mediated vectors.

### Claim 22 (depends on 1)

A genetically engineered cytotoxic cell, comprising a polynucleotide sequence encoding the chimeric antigen receptor of claim 1.

### Claim 23

A method for producing a chimeric antigen receptor comprising at least one antigen specific targeting region, a transmembrane domain and an intracellular signaling domain, said method comprising the steps of
generating the at least one antigen specific targeting region from a parent or wild-type protein or a domain thereof that binds specifically with a tumor specific target antigen, by:
i. evolving the DNA which encodes the parent or wild-type protein or a domain thereof using one or more evolutionary techniques to create mutant DNAs;
ii. expressing the mutant DNAs to obtain mutant polypeptides;
iii. subjecting the mutant polypeptides to an assay under a normal physiological condition and to an assay under an aberrant condition that deviates from the normal physiological condition; and
iv. selecting the at least one antigen specific targeting region from the mutant polypeptides expressed in step (ii) which exhibits a decrease in activity in the assay at the normal physiological condition compared to the activity in the assay under the aberrant condition;
wherein the tumor specific target antigen is selected from the group consisting of tyrosine kinase growth factor receptors that are overexpressed on a surface of a cancer cell.