# US 9,518,978 B2 · Gamma 9 delta 2 T cell receptors

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- Number: US 9,518,978 B2
- Document ID: US9518978B2
- Status: Granted
- Kind: B2
- Application number: 14/388,675
- Filed: 2013-03-28
- Published: 2015-02-19
- Granted: 2016-12-13
- Earliest claimed: 2012-03-28
- Assignees: Gadeta B.V.
- Applicants: UMC UTRECHT Holding B.V.
- Inventors: Jürgen Herbert Ernst Kuball; Elsa-Cordula Gründer
- Web page: https://patentgrep.com/patent/US9518978B2

## CPC

- G01N 33/5011: {for testing antineoplastic activity}
- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C12N 15/85: for animal cells
- G01N 33/505: {involving T-cells}
- G01N 33/6866: {Interferon}
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The current invention provides methods to identify γ9δ2T-cell receptors (γ9δ2TCR) that mediate anti-tumor responses. Surprisingly, it was now found that the CDR3 regions of the γ9-T-cell receptor chain and the δ2-T-Cell receptor chain (δ2TCR chain) are of importance. Based on these findings, combinatorial-γδTCR-chain-exchange (CTE) is proposed as an efficient method for identifying γ9δ2TCRs that mediate anti-tumor responses. Using the method of the invention, specific sequences of the respective γ9TCR and δ2TCR chains were identified that mediate anti-tumor responses. Hence, the invention further provides for specific γ9δ2TCRs, or fragments thereof, that may be used e.g. in diagnostics or treatment of cancer. The invention further provides for nucleic acid sequences, genetic constructs and retroviral vectors that can be used to express the γ9δ2TCRs according to the invention.

## Claims

### Claim 1

A γ9δ2T-cell receptor, or a fragment thereof, comprising a γ9-T-cell receptor chain comprising a γ9-CDR3 region, wherein the amino acid sequence of the γ9-CDR3 region corresponding to amino acid residues 122-124 of SEQ ID NO. 1 is IQ (SEQ ID NO. 20) and a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein the amino acid sequence of the δ2-CDR3 region corresponding to amino acid residues 115-122 of SEQ ID NO. 2 is ALKRTD (SEQ ID NO. 15).

### Claim 2

A γ9δ2T-cell receptor, or a fragment thereof, comprising: a γ9-T-cell receptor chain comprising a γ9-CDR3 region, wherein the amino acid sequence of the γ9-CDR3 region corresponds to the amino acid sequence of SEQ ID NO. 16, and a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein the amino acid sequence of the δ2-CDR3 region corresponds to the amino acid sequence of SEQ ID NO. 17.

### Claim 3 (depends on 1)

The γ9δ2T-cell receptor or fragment thereof according to claim 1, wherein the amino acid sequence of the γ9-CDR3 region corresponding to amino acid residues 118-121 of the amino acid sequence of SEQ ID NO. 1 is ALWE, and wherein the amino acid sequence of the γ9-CDR3 region corresponding to amino acid residues 125-132 of the amino acid sequence of SEQ ID NO. 1 is ELGKKIKV, and wherein the amino acid sequence of the δ2-CDR3 region corresponding to amino acid residues 112-114 of the amino acid sequence of SEQ ID NO. 2 is ACD, and wherein the amino acid sequence of the δ2-CDR3 region corresponding to amino acid residues 123-127 of the amino acid sequence of SEQ ID NO. 2 is TDKLI.

### Claim 4 (depends on 1)

The γ9δ2T-cell receptor, or fragment thereof, according to claim 1, wherein the γ9-T-cell receptor chain comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 1, and wherein the δ2-T-cell receptor chain comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 2.

### Claim 5 (depends on 1)

The γ9δ2T-cell receptor, or fragment thereof, according to claim 1, wherein the γ9-T-cell receptor chain comprises the amino acid sequence of SEQ ID NO. 12 and wherein the δ2-T-cell receptor chain comprises the amino acid sequence of SEQ ID NO. 14.

### Claim 6 (depends on 1)

A conjugate comprising a soluble fragment of the γ9δ2T-cell receptor according to claim 1, comprising said γ9-CDR3 region and said δ2-T-cell δ2-CDR3 region, linked to an agent.

### Claim 7 (depends on 6)

The conjugate according to claim 6, wherein the agent is selected from the group consisting of a diagnostic agent, a therapeutic agent, an anti-cancer agent, a chemical, a nanoparticle, a chemotherapeutic agent and a fluorochrome.

### Claim 8 (depends on 1)

A T cell comprising the γ9δ2T-cell receptor according to claim 1.

### Claim 9 (depends on 8)

A T cell according to claim 8, wherein the T cell comprises:
a) an isolated nucleic acid sequence or isolated nucleic acid sequences encoding:
i) a δ2-T-cell receptor chain, or fragment thereof, comprising a δ2-CDR3 region, wherein the amino acid sequence of the δ2-CDR3 region corresponding to amino acid residues 115-122 of the amino acid sequence of SEQ ID NO. 2 is ALKRTD (SEQ ID NO. 15); and
ii) a γ9-T-cell receptor chain, or fragment thereof, comprising a γ9-CDR3 region, wherein the amino acid sequence of the γ9-CDR3 region corresponding to amino acid residues 122-124 of SEQ ID NO. 1 is IQ (SEQ ID NO. 20),
b) a genetic construct or genetic constructs comprising the isolated nucleic acid sequence(s) or
c) a retroviral vector or retroviral vectors comprising the genetic construct(s).

### Claim 10

A medicament comprising one selected from the group consisting of:
a) γ9δ2T-cell receptor, or fragment thereof, comprising:
i) a γ9-T-cell receptor chain comprising a γ9-CDR3 region, wherein the amino acid sequence of the γ9-CDR3 region corresponding to amino acid residues 122-124 of SEQ ID NO. 1 is IQ (SEQ ID NO. 20) and a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein the amino acid sequence of the δ2-CDR3 region corresponding to amino acid residues 115-122 of SEQ ID NO. 2 is ALKRTD (SEQ ID NO. 15); or
ii) a γ9-T-cell receptor chain comprising a γ9-CDR3 region, wherein the amino acid sequence of the γ9-CDR3 region corresponds to SEQ ID NO. 16 and a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein the amino acid sequence of the δ2-CDR3 region corresponds to SEQ ID NO. 17;
b) a conjugate comprising a soluble fragment of the γ9δ2T-cell receptor, comprising the γ9-CDR3 and the δ2-T-cell δ2-CDR3 region, linked to an agent;
c) an isolated nucleic acid sequence or isolated nucleic acid sequences encoding:
i) the δ2-T-cell receptor chain, or fragment thereof, wherein the δ2-CDR3 region corresponding to amino acid residues 115-122 of SEQ ID NO. 2 is ALKRTD (SEQ ID NO. 15),
ii) the γ9-T-cell receptor chain, or fragment thereof, wherein the amino acid sequence of the γ9-CDR3 region corresponding to amino acid residues 122-124 of the amino acid sequence of SEQ ID NO. 1 is IQ (SEQ ID NO. 20), or
iii) the γ9δ2T-cell receptor, or fragment thereof;
d) a genetic construct or genetic constructs comprising the isolated nucleic acid sequence(s);
e) a retroviral vector or retroviral vectors comprising the genetic construct(s); and
f) a T cell comprising the γ9δ2T-cell receptor.

### Claim 11 (depends on 10)

The medicament according to claim 10, wherein the medicament is useful for the treatment of cancer.

### Claim 12

A γ9δ2T-cell receptor, or a fragment thereof, comprising a γ9-T-cell receptor chain comprising a γ9-CDR3 region, wherein the amino acid sequence of the γ9-CDR3 region corresponding to amino acid residues 122-124 of SEQ ID NO. 1 is modified and a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein the amino acid sequence of the δ2-CDR3 region corresponding to amino acid residues 115-122 of SEQ ID NO. 2 is modified, wherein the combinations of the amino acid modifications within the γ9δ2T-cell receptor, or a fragment thereof, are as listed in Table 1.

### Claim 13 (depends on 12)

A γ9δ2T-cell receptor, or a fragment thereof, comprising: a γ9-T-cell receptor chain comprising a γ9-CDR3 region according to claim 12, wherein the amino acid sequence of the γ9-CDR3 region corresponding to amino acid residues 122-124 of the amino acid sequence of SEQ ID NO. 1 is AQQ (SEQ ID NO. 21), and a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein the amino acid sequence of the δ2-CDR3 region corresponding to amino acid residues 115-122 of the amino acid sequence of SEQ ID NO. 2 is ALKRTD (SEQ ID NO. 15).

### Claim 14 (depends on 12)

A γ9δ2T-cell receptor, or a fragment thereof, comprising: a γ9-T-cell receptor chain comprising a γ9-CDR3 region according to claim 12, wherein the amino acid sequence of the γ9-CDR3 region corresponding to amino acid residues 122-124 of the amino acid sequence of SEQ ID NO. 1 is IQ (SEQ ID NO. 20), and a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein the amino acid sequence of the δ2-CDR3 region corresponding to amino acid residues 115-122 of the amino acid sequence of SEQ ID NO. 2 is TLGMGGEY (SEQ ID NO. 19).

### Claim 15 (depends on 12)

A γ9δ2T-cell receptor, or a fragment thereof, comprising: a γ9-T-cell receptor chain comprising a γ9-CDR3 region according to claim 12, wherein the amino acid sequence of the γ9-CDR3 region corresponding to amino acid residues 122-124 of the amino acid sequence of SEQ ID NO. 1 is AQQ (SEQ ID NO. 21), and a δ2-T-cell receptor chain comprising a δ2-CDR3 region wherein the amino acid sequence of the δ2-CDR3 region corresponding to amino acid residues 115-122 of the amino acid sequence of SEQ ID NO. 2 is LLGY (SEQ ID NO. 22).

### Claim 16 (depends on 12)

A γ9δ2T-cell receptor, or fragment thereof, according to claim 12, wherein the γ9-T-cell receptor chain comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 1, and wherein the δ2-T-cell receptor chain comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 2, wherein the amino acid sequence of the γ9-CDR3 region corresponding to amino acid residues number 122-124 of SEQ ID NO. 1, and the amino acid sequence of the δ2-CDR3 region corresponding to amino acid residues 115-122 of SEQ ID NO. 2 are as listed in Table 1.