IMMUNOTHERAPY CONSTRUCTS TARGETING KRAS ANTIGENS
20220227883 · 2022-07-21
Inventors
Cpc classification
C07K2317/32
CHEMISTRY; METALLURGY
A61K35/17
HUMAN NECESSITIES
C12N5/0638
CHEMISTRY; METALLURGY
G01N33/57492
PHYSICS
C07K2317/34
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61P35/00
HUMAN NECESSITIES
G01N33/50
PHYSICS
Abstract
An antigen targeting agent is provided. The antigen targeting agent binds to a mutated Kirsten rat sarcoma viral oncogene homolog (KRAS) protein having a missense mutation at position 12 when a peptide incorporating the missense mutation is presented by an HLA-A*02 molecule. The missense mutation at position 12 of the KRAS protein may be G12D, G12V or G12C. The antigen targeting agents can be used diagnostically or for immunotherapy.
Claims
1. An antigen targeting agent comprising an antigen binding site that binds to a mutated Kirsten rat sarcoma viral oncogene homolog (KRAS) protein having a missense mutation at position 12 when a peptide incorporating the missense mutation is presented by an HLA-A*02 molecule.
2. An antigen targeting agent as defined in claim 1, wherein the missense mutation at position 12 of the KRAS protein is G12D, G12V or G12C.
3. An antigen targeting agent as defined in claim 1, wherein the HLA-A*02 molecule is HLA-A*02:01.
4. An antigen targeting agent as defined in claim 1, wherein: the missense mutation at position 12 of the KRAS protein is G12V, and wherein the HLA-A*02 molecule is an HLA-A02:253, HLA-A02:03, HLA-A02:264, HLA-A02:258, HLA-A02:230, HLA-A02:69, HLA-A02:11, HLA-A02:128, HLA-A02:104, HLA-A02:22, HLA-A02:50, HLA-A02:26, HLA-A02:171, HLA-A02:141, HLA-A02:99, HLA-A02:13, HLA-A02:90, HLA-A02:158, HLA-A02:131, HLA-A02:16, HLA-A02:102, HLA-A02:155, HLA-A02:63, HLA-A02:02, HLA-A02:186, HLA-A02:115, HLA-A02:209, HLA-A02:47, HLA-A02:29, HLA-A02:263, HLA-A02:116, HLA-A02:241, HLA-A02:71, HLA-A02:59, HLA-A02:40, HLA-A02:166, HLA-A02:238, HLA-A02:176, HLA-A02:75, HLA-A02:30, HLA-A02:174, HLA-A02:266, HLA-A02:187, HLA-A02:85, HLA-A02:165, HLA-A02:160, HLA-A02:183, HLA-A02:189, HLA-A02:138, HLA-A02:228, HLA-A02:260, HLA-A02:107, HLA-A02:215, HLA-A02:182, HLA-A02:09, HLA-A02:192, HLA-A02:163, HLA-A02:221, HLA-A02:159, HLA-A02:194, HLA-A02:140, HLA-A02:206, HLA-A02:74, HLA-A02:198, HLA-A02:123, HLA-A02:95, HLA-A02:168, HLA-A02:150, HLA-A02:210, HLA-A02:86, HLA-A02:235, HLA-A02:237, HLA-A02:208, HLA-A02:212, HLA-A02:201, HLA-A02:120, HLA-A02:240, HLA-A02:211, HLA-A02:175, HLA-A02:162, HLA-A02:121, HLA-A02:89, HLA-A02:220, HLA-A02:164, HLA-A02:190, HLA-A02:157, HLA-A02:96, HLA-A02:256, HLA-A02:234, HLA-A02:97, HLA-A02:204, HLA-A02:70, HLA-A02:77, HLA-A02:93, HLA-A02:181, HLA-A02:111, HLA-A02:118, HLA-A02:196, HLA-A02:185, HLA-A02:214, HLA-A02:193, HLA-A02:200, HLA-A02:25, HLA-A02:173, HLA-A02:177, HLA-A02:207, HLA-A02:257, HLA-A02:203, HLA-A02:199, HLA-A02:66, HLA-A02:01, HLA-A02:216, HLA-A02:133, HLA-A02:119, HLA-A02:153, HLA-A02:251, HLA-A02:145, HLA-A02:24, HLA-A02:197, HLA-A02:236, HLA-A02:149, HLA-A02:68, HLA-A02:218, HLA-A02:205, HLA-A02:31, HLA-A02:239, HLA-A02:109, HLA-A02:67, HLA-A02:132, HLA-A02:134, HLA-A02:252, HLA-A02:202, HLA-A02:213, HLA-A02:35, HLA-A02:161, HLA-A02:245, HLA-A02:73, HLA-A02:105, HLA-A02:12, HLA-A02:27, HLA-A02:148, HLA-A02:139, HLA-A02:78, HLA-A02:262, HLA-A02:38, HLA-A02:41, HLA-A02:167, HLA-A02:58, HLA-A02:34, HLA-A02:20, HLA-A02:233, HLA-A02:147, HLA-A02:151, HLA-A02:42, HLA-A02:60, HLA-A02:62, HLA-A02:126, HLA-A02:51, HLA-A02:61, HLA-A02:79, HLA-A02:137, HLA-A02:170, HLA-A02:06, HLA-A02:28, HLA-A02:72, HLA-A02:259, HLA-A02:180, HLA-A02:91, HLA-A02:248, HLA-A02:106, HLA-A02:144, HLA-A02:21, HLA-A02:44, HLA-A02:142, HLA-A02:122, HLA-A02:48, HLA-A02:127, HLA-A02:52, HLA-A02:254, HLA-A02:243, HLA-A02:224, HLA-A02:36, HLA-A02:169, or HLA-A02:101 molecule; the missense mutation at position 12 of the KRAS protein is G12D, and wherein the HLA-A*02 molecule is an HLA-A02:03, HLA-A02:253, HLA-A02:230, HLA-A02:258, HLA-A02:264, HLA-A02:11, HLA-A02:69, HLA-A02:128, HLA-A02:22, HLA-A02:104, HLA-A02:50, HLA-A02:26, HLA-A02:171, HLA-A02:99, HLA-A02:13, HLA-A02:02, HLA-A02:63, HLA-A02:102, HLA-A02:115, HLA-A02:209, HLA-A02:155, HLA-A02:186, HLA-A02:141, HLA-A02:90, HLA-A02:47, HLA-A02:158, HLA-A02:16, HLA-A02:131, HLA-A02:148, HLA-A02:263, HLA-A02:29, HLA-A02:12, HLA-A02:116, HLA-A02:27, HLA-A02:105, HLA-A02:73, HLA-A02:245, HLA-A02:01, HLA-A02:09, HLA-A02:31, HLA-A02:40, HLA-A02:24, HLA-A02:25, HLA-A02:30, HLA-A02:59, HLA-A02:66, HLA-A02:67, HLA-A02:68, HLA-A02:70, HLA-A02:71, HLA-A02:74, HLA-A02:75, HLA-A02:77, HLA-A02:85, HLA-A02:86, HLA-A02:89, HLA-A02:93, HLA-A02:95, HLA-A02:96, HLA-A02:97, HLA-A02:107, HLA-A02:109, HLA-A02:111, HLA-A02:118, HLA-A02:119, HLA-A02:120, HLA-A02:173, HLA-A02:174, HLA-A02:175, HLA-A02:176, HLA-A02:177, HLA-A02:181, HLA-A02:212, HLA-A02:213, HLA-A02:214, HLA-A02:215, HLA-A02:216, HLA-A02:218, HLA-A02:220, HLA-A02:221, HLA-A02:202, HLA-A02:203, HLA-A02:204, HLA-A02:205, HLA-A02:206, HLA-A02:207, HLA-A02:208, HLA-A02:210, HLA-A02:211, HLA-A02:237, HLA-A02:238, HLA-A02:239, HLA-A02:240, HLA-A02:241, HLA-A02:132, HLA-A02:133, HLA-A02:134, HLA-A02:138, HLA-A02:140, HLA-A02:153, HLA-A02:157, HLA-A02:159, HLA-A02:160, HLA-A02:162, HLA-A02:163, HLA-A02:164, HLA-A02:165, HLA-A02:166, HLA-A02:168, HLA-A02:251, HLA-A02:252, HLA-A02:256, HLA-A02:257, HLA-A02:145, HLA-A02:149, HLA-A02:150, HLA-A02:192, HLA-A02:193, HLA-A02:194, HLA-A02:196, HLA-A02:197, HLA-A02:198, HLA-A02:199, HLA-A02:200, HLA-A02:201, HLA-A02:228, HLA-A02:234, HLA-A02:235, HLA-A02:236, HLA-A02:260, HLA-A02:266, HLA-A02:182, HLA-A02:183, HLA-A02:185, HLA-A02:187, HLA-A02:189, HLA-A02:190, HLA-A02:121, HLA-A02:123, HLA-A02:161, HLA-A02:35, HLA-A02:38, HLA-A02:139, HLA-A02:262, HLA-A02:41, HLA-A02:58, HLA-A02:233, HLA-A02:147, HLA-A02:151, HLA-A02:167, HLA-A02:20, HLA-A02:122, HLA-A02:44, HLA-A02:142, HLA-A02:34, HLA-A02:42, HLA-A02:78, HLA-A02:06, HLA-A02:21, HLA-A02:28, HLA-A02:51, HLA-A02:61, HLA-A02:72, HLA-A02:79, HLA-A02:91, HLA-A02:106, HLA-A02:180, HLA-A02:137, HLA-A02:170, HLA-A02:248, HLA-A02:144, HLA-A02:259, HLA-A02:126, HLA-A02:243, HLA-A02:52, HLA-A02:48, HLA-A02:60, HLA-A02:62, HLA-A02:127, or HLA-A02:229 molecule; or the missense mutation at position 12 of the KRAS protein is G12C, and wherein the HLA-A*02 molecule is an HLA-A02:253, HLA-A02:03, HLA-A02:264, HLA-A02:258, HLA-A02:230, HLA-A02:69, HLA-A02:11, HLA-A02:104, HLA-A02:22, HLA-A02:50, HLA-A02:128, HLA-A02:26, HLA-A02:171, HLA-A02:99, HLA-A02:102, HLA-A02:155, HLA-A02:63, HLA-A02:02, HLA-A02:186, HLA-A02:115, HLA-A02:209, HLA-A02:47, HLA-A02:13, HLA-A02:141, HLA-A02:90, HLA-A02:148, HLA-A02:158, HLA-A02:131, HLA-A02:16, HLA-A02:263, HLA-A02:116, HLA-A02:29, HLA-A02:35, HLA-A02:38, HLA-A02:105, HLA-A02:12, HLA-A02:245, HLA-A02:73, HLA-A02:241, HLA-A02:71, HLA-A02:59, HLA-A02:40, HLA-A02:166, HLA-A02:238, HLA-A02:176, HLA-A02:75, HLA-A02:30, HLA-A02:174, HLA-A02:266, HLA-A02:187, HLA-A02:85, HLA-A02:165, HLA-A02:160, HLA-A02:183, HLA-A02:189, HLA-A02:138, HLA-A02:228, HLA-A02:260, HLA-A02:107, HLA-A02:215, HLA-A02:182, HLA-A02:09, HLA-A02:192, HLA-A02:163, HLA-A02:221, HLA-A02:159, HLA-A02:194, HLA-A02:140, HLA-A02:206, HLA-A02:74, HLA-A02:198, HLA-A02:123, HLA-A02:95, HLA-A02:168, HLA-A02:150, HLA-A02:210, HLA-A02:86, HLA-A02:235, HLA-A02:237, HLA-A02:208, HLA-A02:212, HLA-A02:201, HLA-A02:120, HLA-A02:240, HLA-A02:211, HLA-A02:175, HLA-A02:162, HLA-A02:121, HLA-A02:89, HLA-A02:220, HLA-A02:164, HLA-A02:190, HLA-A02:157, HLA-A02:96, HLA-A02:256, HLA-A02:234, HLA-A02:97, HLA-A02:204, HLA-A02:70, HLA-A02:77, HLA-A02:93, HLA-A02:181, HLA-A02:111, HLA-A02:118, HLA-A02:196, HLA-A02:185, HLA-A02:214, HLA-A02:193, HLA-A02:200, HLA-A02:25, HLA-A02:173, HLA-A02:177, HLA-A02:207, HLA-A02:257, HLA-A02:203, HLA-A02:199, HLA-A02:66, HLA-A02:01, HLA-A02:216, HLA-A02:133, HLA-A02:119, HLA-A02:153, HLA-A02:251, HLA-A02:145, HLA-A02:24, HLA-A02:197, HLA-A02:236, HLA-A02:149, HLA-A02:68, HLA-A02:218, HLA-A02:205, HLA-A02:31, HLA-A02:239, HLA-A02:109, HLA-A02:67, HLA-A02:132, HLA-A02:134, HLA-A02:252, HLA-A02:202, HLA-A02:213, HLA-A02:161, HLA-A02:122, HLA-A02:27, HLA-A02:262, HLA-A02:233, HLA-A02:41, HLA-A02:139, HLA-A02:44, HLA-A02:142, HLA-A02:58, HLA-A02:229, HLA-A02:167, HLA-A02:147, or HLA-A02:151 molecule.
5. (canceled)
6. (canceled)
7. An antigen targeting agent as defined in claim 1, the agent comprising first and second chains, each one of the first and second chains having first, second and third complementarity determining regions (CDRs), wherein: the third CDR of the first chain comprises the amino acid sequence of SEQ ID NO:30 or SEQ ID NO:34, and wherein the third CDR of the second chain comprises the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:36; the first chain comprises the amino acid sequence of TRAV27*01 (SEQ ID NO:6) or the amino acid sequence of TRAV13-2*01 (SEQ ID NO:10); the second chain comprises the amino acid sequence of TRBV 19*01 (SEQ ID NO:8) or the amino acid sequence of TRBV 04-1*01 (SEQ ID NO:12); the first CDR of the first chain comprises SEQ ID NO:14 or SEQ ID NO:18; the second CDR of the first chain comprises SEQ ID NO:16 or SEQ ID NO:20; the first CDR of the second chain comprises SEQ ID NO:22 or SEQ ID NO:26; the first CDR of the second chain comprises SEQ ID NO:22 or SEQ ID NO:26; and/or the second CDR of the second chain comprises SEQ ID NO:24 or SEQ ID NO:28.
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. An antigen targeting agent as defined in claim 1, wherein: the first chain comprises as its first, second and third CDRs SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:30, respectively, and the second chain comprises as its first, second and third CDRs SEQ ID NO:22, SEQ ID NO:26 and SEQ ID NO:32, respectively; the first chain comprises as its first, second and third CDRs SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:34, respectively, and the second chain comprises as its first, second and third CDRs SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:32, respectively; the first chain comprises as its first, second and third CDRs SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:30, respectively, and the second chain comprises as its first, second and third CDRs SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:36, respectively; or the first chain comprises as its first, second and third CDRs SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:34, respectively, and the second chain comprises as its first, second and third CDRs SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:36, respectively.
15. An antigen targeting agent as defined in claim 1, wherein: the missense mutation at position 12 of the KRAS is G12V, and the third CDR of the first chain has the amino acid sequence of SEQ ID NO:30 and the third CDR of the second chain has the amino acid sequence of SEQ ID NO:32; the missense mutation at position 12 of the KRAS is G12D, and the third CDR of the first chain has the amino acid sequence of SEQ ID NO:34 and the third CDR of the second chain has the amino acid sequence of SEQ ID NO:32; or the missense mutation at position 12 of the KRAS is G12D, and the third CDR of the first chain has the amino acid sequence of SEQ ID NO:30 and the third CDR of the second chain has the amino acid sequence of SEQ ID NO:36.
16. An antigen targeting agent as defined in claim 1, wherein: the first and second chains of the antigen targeting agent comprise a single polypeptide, or wherein the first and second chains of the antigen targeting agent comprise two separate polypeptides; the first and second chains of the antigen targeting agent are configured to be expressed as a single polypeptide with a suitable sequence interposing the first and second chains so that the first and second chains are cleaved into or translated as two separate polypeptides in vivo, wherein the suitable sequence optionally comprises a T2A, P2A, E2A, F2A or IRES sequence.
17. (canceled)
18. An antigen targeting agent as defined in claim 1, wherein the antigen targeting agent comprises a T-cell receptor (TCR), wherein optionally: the first chain comprises an alpha-chain of the TCR, and the second chain comprises a beta-chain of the TCR; the first chain comprises a gamma-chain of the TCR, and the second chain comprises a delta-chain of the TCR; constant regions of the TCR comprise murine constant regions; and/or the T-cell receptor comprises the amino acid sequence of any one of SEQ ID NOs:38, 40, 42 or 44.
19. (canceled)
20. (canceled)
21. (canceled)
22. An antigen targeting agent as defined in claim 1, wherein the antigen targeting agent comprises a chimeric antigen receptor (CAR), and wherein the three complementarity determining regions of each of the first and second chains are configured to be expressed as a single polypeptide together with a co-stimulatory domain; or wherein the antigen targeting agent comprises a bi-specific antibody, the bi-specific antibody having a first domain comprising the antigen-binding site that binds to the KRAS protein having a missense mutation at position 12 when the peptide incorporating the missense mutation is presented by an HLA-A*02 molecule, and a second domain comprising an antigen binding site configured to recruit cytotoxic cells, optionally wherein the second domain of the bi-specific antibody binds CD3.
23. (canceled)
24. (canceled)
25. An antigen targeting agent as defined in claim 1, wherein the antigen targeting agent specifically binds to the peptide incorporating the missense mutation at position 12 of the KRAS protein when the peptide is presented by an HLA-A*02 molecule; or wherein the antigen targeting agent is expressed by a cell that has been genetically engineered to express the antigen targeting agent.
26. (canceled)
27. (canceled)
28. An isolated or purified antigen targeting agent as defined in claim 1.
29. An isolated nucleic acid molecule having a DNA sequence encoding an antigen targeting agent as defined in claim 1.
30. An isolated nucleic acid molecule as defined in claim 29 having the nucleotide sequence of any one of SEQ ID NOs:37, 39, 41, 43, 45, 46, 47 or 48.
31. A pharmaceutical composition comprising an antigen targeting agent as defined in claim 1 and a pharmaceutically acceptable carrier.
32. A cytotoxic cell that has been genetically engineered to express an antigen targeting agent as defined in claim 1.
33. A cytotoxic cell comprising a nucleic acid molecule as defined in claim 30.
34. A cytotoxic cell as defined in claim 32, wherein the cytotoxic cell is a CD8.sup.+ T-cell, CD4.sup.+ T-cell or natural killer cell.
35. A method of producing a cytotoxic cell capable of expressing an antigen targeting agent to bind KRAS peptides having a missense mutation at position 12 as presented by HLA-A*02 molecules, the method comprising: obtaining cytotoxic cells from a source; and genetically engineering the cytotoxic cells using a nucleotide vector comprising the nucleic acid molecule of claim 29.
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
41. (canceled)
42. (canceled)
43. (canceled)
44. A method of detection of cancer in a mammalian subject, the method comprising: contacting a sample comprising cells obtained from the subject with an antigen targeting agent or a cytotoxic cell as defined in claim 1; if the cells express KRAS.sup.G12X antigens, the antigen targeting agent or the cytotoxic cell binds to the KRAS.sup.G12X antigens, thereby forming a complex; and detecting the presence of the complex, wherein the presence of the complex is indicative of cancer in the mammal; or the method comprising: obtaining a sample from the subject; co-culturing cells from the sample with cytotoxic cells capable of binding to KRAS.sup.G12X peptides as displayed by HLA-A*02 molecules, wherein the cytotoxic cells express an antigen targeting agent as defined in claim 1; and evaluating an indicator of cytotoxic activity; wherein a presence of or increase in a level of the indicator of cytotoxic activity indicates a cancer involving a missense mutation at position 12 of KRAS.
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
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DESCRIPTION
[0076] Throughout the following description specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.
[0077] As used herein, the terms “CD8.sup.+ T-cells” and “TCD8.sup.+” refer to CD8-positive T-cells. CD8-positive T-cells are able recognize and destroy cells flagged by MHC class I molecules and this ability is known as MHC class I-restriction. CD8-positive T-cells include cytotoxic T-cells (CTLs). Similarly, “CD4.sup.+ T-cells” refers to CD4-positive T-cells.
[0078] As used herein, the term “antigen” refers to molecules that can induce an immune response. For example, an antigen may be one that is recognisable by cytotoxic T-cells to stimulate an anti-tumour immune response.
[0079] As used herein, the term “epitope” refers to the part of an antigen that can stimulate an immune response. For example, an epitope may be a peptide that is bound to a MHC class I molecule to thereby form a MHC/peptide complex. The MHC/peptide complex can be selectively recognized by a suitable T-cell receptor of a cytotoxic T-cell to stimulate an anti-tumour immune response.
[0080] As used herein, the term “DNA” refers to deoxyribonucleic acid. The information stored in DNA is coded as a sequence made up generally of four chemical bases: adenine (A), guanine (G), cytosine (C) and thymine (T). Other bases and chemically modified bases exist as well and are encompassed within certain embodiments. As used herein, reference to a DNA sequence includes both single and double stranded DNA. A specific sequence refers to (i) a single stranded DNA of such sequence, (ii) a double stranded DNA comprising a single stranded DNA of such sequence and its complement, and (iii) the complement of such sequence.
[0081] As used herein, the term “fragment” means a portion of a larger whole. In the context of a DNA coding sequence, a fragment means a portion of the DNA sequence that is less than the complete coding region. However, the expression product of the fragment may retain substantially the same biological function as the expression product of the complete coding sequence.
[0082] As used herein, the term “peptide” means a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acid. A peptide may be immunogenic, meaning that the peptide is capable of inducing an immune response, e.g. a T-cell response.
[0083] As used herein, the term “isolated” means that a material is separated/removed from its original environment. For example, HLA-A*02:01:KRAS.sup.G12D&V-reactive CD8.sup.+ T cells removed from their natural environment, e.g. blood, are isolated. HLA-A*02:01:KRAS.sup.G12D&V-reactive CD8.sup.+ T cells present their natural environment within a pancreatic cancer patient are not isolated.
[0084] As used herein, the term “purified” does not mean absolute purity. Instead, it can include preparations that undergo a purification process, e.g. highly purified preparations and partially purified preparations having a purity of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% pure.
[0085] As used herein, the term “T-cell response” means the proliferation and activation of effector T-cells. For example, T-cell response of MHC class I restricted cytotoxic T-cells may include lysis of target cells, secretion of cytokines, and secretion of effector molecules (e.g. perforins and granzymes).
[0086] As used herein, the term “variant” means in the context of proteins, one or two or more of the amino acid residues are replaced with other amino acid residues, while the variant retains substantially the same biological function as the unaltered protein.
[0087] The terms “treat”, “treating” and “treatment” refer to an approach for obtaining desired clinical results. Desired clinical results can include, but are not limited to, reduction or alleviation of at least one symptom of a disease. For example, treatment can be diminishment of at least one symptom of disease, diminishment of extent of disease, stabilization of disease state, prevention of spread of disease, delay or slowing of disease progression, palliation of disease, diminishment of disease reoccurrence, remission of disease, prolonging survival with disease, or complete eradication of disease.
[0088] The terms “cancer cell” and “tumor cell” refer to cells, the growth and division of which can be typically characterized as unregulated. Cancer cells can be of any origin, including benign and malignant cancers, metastatic and non-metastatic cancers, and primary and secondary cancers.
[0089] As used herein, the term “KRAS.sup.G12X” refers to KRAS missense mutants at KRAS codon position 12. As used herein, the term “KRAS.sup.G12D&V” refers to KRAS.sup.G12D and KRAS.sup.G12V mutant KRAS, i.e. KRAS having a missense mutation at position 12 wherein the wild type glycine residue is mutated to an aspartic acid residue or a valine, respectively. “KRAS.sup.G12C” refers to KRAS in which the wild type glycine residue at position 12 is mutated to a cysteine residue.
[0090] In one embodiment, the inventors have discovered an antigen targeting receptor targeting KRAS.sup.G12X antigens/mutants that can be used to stimulate anti-tumour immune responses. In some embodiments, the antigen targeting receptor is a T-cell receptor. The T-cell receptor is engineered to recognize and bind to KRAS.sup.G12X antigens/mutant peptides that are presented by MHC class I molecules of the subclass HLA-A*02:01. Because many cancer cells express KRAS.sup.G12X antigens/mutants and because HLA-A*02:01 is a highly prevalent HLA-A subtype, the novel antigen targeting receptor of some embodiments can be used for cancer screening, treatment and prevention in a large segment of the patient population. For example, cytotoxic cells such as CD8.sup.+ T cells may be engineered to express the novel antigen targeting receptors, e.g. as T-cell receptors (TCRs) or chimeric antigen receptors (CARs). When the TCRs or CARs recognize and bind to KRAS.sup.G12X antigens expressed on tumour cells and presented by HLA-A*02:01, CD8+ T cells are activated and can kill the tumour cells, e.g. through lysis of the tumour cells, secretion of cytokines, and/or secretion of effector molecules (e.g. perforins and granzymes).
Antigen Targeting Agents
[0091] Some embodiments of the present invention relate to antigen targeting agents, including antigen targeting receptors. These antigen targeting agents are configured to target KRAS.sup.G12X antigens presented by HLA-A*02 molecules to stimulate anti-tumour immune responses, for example by positioning cytotoxic cells such as T-cells adjacent tumour cells to promote killing of the tumour cells by the cytotoxic cells. In some embodiments, these antigen targeting agents are configured to target KRAS.sup.G12X antigens presented by HLA-A*02:01 molecules.
[0092] In some embodiments, these antigen targeting agents are specific for KRAS.sup.G12X antigens as displayed by HLA-A*02 molecules, meaning that the agents can specifically bind to and immunologically recognize KRAS.sup.G12X antigens with high avidity. For example, an antigen targeting agent may be considered to have antigenic specificity for KRAS.sup.G12X antigens if T cells expressing a TCR incorporating the antigen targeting agent secrete at least twice as much IFNγ upon co-culture with HLA-A*02:01 positive antigen presenting cells (APC) (e.g. K562b cells modified to express HLA-A*02:01) pulsed with the KRAS.sup.G12X peptide having a relevant target mutation at position 12 of KRAS as compared to the amount of IFNγ expressed by a negative control. IFNγ secretion may be measured by methods known in the art such as, for example, enzyme-linked immunosorbent assay (ELISA).
[0093] In some embodiments, the targeted KRAS.sup.G12X antigens are KRAS.sup.G12D/V/C antigens. Wild type KRAS (KRAS.sup.WT) contains a ten amino acid fragment having the sequence KLVVVGAGGV (SEQ ID NO:1). In some embodiments, the targeted KRAS.sup.G12DN antigens have the amino acid sequences set forth in SEQ ID NO:2 (KLVVVGAVGV, a peptide corresponding KRAS having a missense mutation at position 12 of G12V, referred to herein as KRAS.sup.G12V) and SEQ ID NO:3 (KLVVVGADGV, a peptide corresponding to KRAS having a missense mutation at position 12 of G12D, referred to herein as KRAS.sup.G12D). In some embodiments, the targeted KRASGI2X antigens are KRAS.sup.G12C antigens having the amino acid sequence set forth in SEQ ID NO:4 (KLVVVGACGV, a peptide corresponding to KRAS having a missense mutation at position 12 of G12C).
[0094] In some embodiments, the targeted KRAS.sup.G12X antigens are variants of SEQ ID NOs:2-4 or other peptides incorporating a missense mutation at position 12 of KRAS that vary in length, e.g. that contain one, two, three, four or five additional amino acids from the KRAS protein at the N-terminus and/or at the C-terminus of the peptide, and/or which contain one, two or three fewer amino acids from the KRAS protein at the N-terminus and/or one or two fewer amino acids at the C-terminus of the peptide. In some embodiments, the targeted antigens have additional amino acids at the N-terminal and/or C-terminal end of the peptide, e.g. one, two, three, four or five additional amino acids at the N-terminus of the peptide, and/or one, two, three, four or five additional amino acids at the C-terminus of the peptide. In some embodiments, the targeted antigens have fewer amino acids at the N-terminal and/or C-terminal end of the peptide e.g. with one, two or three amino acids removed from the KRAS protein at the N-terminus and/or one or two amino acids removed at the C-terminus of the peptide. In some embodiments, the targeted KRAS.sup.G12X antigens are 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer or 16-mer peptides incorporating the missense mutation at position 12 of KRAS.
[0095] In some embodiments, the antigen targeting agents have an antigen binding site that is specific for KRAS.sup.G12X antigens presented at the cell surface by HLA-A*02 molecules. In some embodiments, the HLA-A*02 molecules are HLA-A*02:01 molecules.
[0096] In some embodiments, the antigen targeting agents target cytotoxic cells to tumour cells. For example, in some embodiments, the antigen targeting agent is a T-cell receptor (TCR) that targets a T-cell incorporating the construct to tumour cells expressing the target missense mutation at position 12 of KRAS. In some embodiments, the antigen targeting agent is a chimeric antigen receptor (CAR) that targets a cytotoxic cell such as a T-cell to tumour cells expressing the target missense mutation at position 12 of KRAS. In some embodiments, the antigen targeting agent is an agent such as a bi-specific antibody that has a first antigen-binding domain that binds to a target KRAS.sup.G12X antigen as presented by HLA-A*02 molecules to target the agent to tumour cells and a second antigen-binding domain that targets cytotoxic cells, for example that binds to CD3 to target T-cells to the tumour cells.
[0097] Any type of immunotherapy agent that can be used to target cytotoxic cells to tumour cells can be used in various embodiments. In some embodiments, bispecific antibodies that bind to both a KRAS.sup.G12X antigen presented at the cell surface by HLA-A*02 molecules and a factor such as CD3 that can be used to target cytotoxic cells such as T-cells to the tumour cells bound by the bispecific antibody can be used. In some embodiments, an antigen targeting receptor that can be used to conduct cellular immunotherapy can be used. In some embodiments, the antigen targeting receptor is a T-cell receptor (TCR). In some embodiments, the antigen targeting receptor is a chimeric antigen receptor (CAR). In some embodiments, the antigen targeting receptor is a modified form of TCR-CAR construct with a single chain antigen-binding domain of a TCR fused to the signaling domain of a CAR molecule.
[0098] In some embodiments, the antigen targeting agent is a TCR. The TCR has (i) a first chain having first, second and third complementarity-determining regions (CDR1, CDR2, and CDR3) and (ii) a second chain having first, second and third complementarity-determining regions (CDR1, CDR2, and CDR3). In some embodiments, the first and second chains of the TCR are the alpha chain and beta chain, respectively, of a TCR. In some embodiments, the first and second chains of the TCR are the gamma chain and delta chain, respectively, of a TCR. Without being bound by theory, the third complementarity-determining regions (CDR3) are believed to play an important role in KRAS.sup.G12X antigen binding and specificity whereas the first and second complementarity-determining regions (CDR1 and CDR2) are believed to play a role in binding to the MHC Class I backbone (e.g. to the HLA-A*02 molecules). TCR sequences, like antibody sequences, are generated by somatic VDJ recombination and are highly stochastic.
[0099] The design and structure of synthetic TCRs generally is known in the art. In some embodiments, each of the first and second chains of the synthetic TCRs has one or more of the following domains: a hinge domain, a transmembrane domain, and an intracellular T-cell signalling domain. In some embodiments, the intracellular domains of the TCR do not signal directly, but rather form complexes with other molecules such as CD3 subunits that facilitate signalling.
[0100] In some embodiments in which the antigen targeting agent is a T-cell receptor, the antigen targeting agent is expressed from a nucleotide construct capable of expressing both chains of the TCR as a single polypeptide. In some embodiments, the single polypeptide has a linker peptide linking the first and second chains of the T-cell receptor. The linker peptide may facilitate the expression of a recombinant TCR in a host cell.
[0101] In some embodiments, the single polypeptide incorporating both the first and second chains of the synthetic TCR includes a cleavage sequence interposed between the first and second chains of the TCR, so that the first and second chains will be expressed as a single polypeptide and then cleaved into two separate polypeptides in vivo. In some embodiments, the nucleic acid encoding the polypeptide that forms the TCR includes a skipping sequence or a sequence allowing initiation of translation at a site other than the 5′ end of an mRNA molecule, or any other sequence that allows two distinct polypeptides to be translated from a single mRNA, interposed between the nucleic acid encoding the first and second chains of the TCR. Any suitable sequence may be used for this purpose between the first and second chains of the TCR, for example a T2A, P2A, E2A, F2A, or IRES sequence, or the like.
[0102] The order of the first and second chains of the synthetic TCRs in the polynucleotide sequence encoding the TCR and in the resulting polypeptide is interchangeable (i.e in some embodiments, the first chain is provided at the 5′ end of the polynucleotide sequence/the N-terminal direction of the polypeptide, while in other embodiments the second chain is provided at the 5′ end of the polynucleotide sequence/the C-terminal direction of the polypeptide). In some embodiments, the variable domains of the α chain (V.sub.α) and the β chain (V.sub.β) comprise any pairwise combination of the variable regions and/or the CDRs having the amino acid sequences of SEQ ID NOs: 38, 40, 42 and 44.
[0103] In some embodiments, the constant domains of the first and second chains, e.g. the alpha chain (C.sub.α) and the beta chain (C.sub.β) comprise human constant gene segments. In other embodiments, human constant gene segments are replaced with constant gene segments from a different organism, e.g. with murine constant gene segments. An advantage of such replacement is to limit mispairing of the engineered TCR chains, e.g. alpha and beta chains, with the T cell's endogenous T-cell receptor chains, e.g. alpha and beta chains.
[0104] In some embodiments, the constant domains of the first and second chains are further modified in any suitable manner to enhance and/or regulate the interaction therebetween. For example residues of the transmembrane domains of each of the first and second chains that are positioned adjacent to one another in vivo may be changed to cysteine residues, to encourage the formation of additional disulfide bonds between the engineered first and second chains (while such disulfide bonds would not form with endogenous T-cell receptor chains).
[0105] In some embodiments, instead of using TCR constant domains to form a dimer between the first and second chains of the TCR, the synthetic TCRs are provided with any other suitable protein domain that supports dimerization of the two chains, for example a leucine zipper domain.
[0106] In some embodiments, the CDR3 of the alpha chain has the amino acid sequence set forth in SEQ ID NO:30 or the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, the CDR3 of the beta chain has the amino acid sequence set forth in SEQ ID NO:32 or the amino acid sequence set forth in SEQ ID NO:36.
[0107] The first and second complementarity-determining regions (CDR1 and CDR2) can have any amino acid sequences as long as they are configured to engage with KRAS.sup.G12Xpeptides presented by HLA-A*02 molecules, including HLA-A*02:01 molecules. For example, in some embodiments, the CDR1 of the alpha chain has the amino acid sequence set forth in SEQ ID NO:14 or the amino acid sequence set forth in SEQ ID NO:18. In some embodiments, the CDR2 of the alpha chain has the amino acid sequence set forth in SEQ ID NO:16 or the amino acid sequence set forth in SEQ ID NO:20.
[0108] In some embodiments, the CDR1 of the beta chain has the amino acid sequence set forth in SEQ ID NO:22 or the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the CDR2 of the beta chain has the amino acid sequence set forth in SEQ ID NO:24 or the amino acid sequence set forth in SEQ ID NO:28.
[0109] In some embodiments, the TCR has (i) an alpha chain having first, second and third complementarity-determining regions (CDR1, CDR2, and CDR3) having the amino acid sequences set forth in SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:30, respectively; and (ii) a beta chain having first, second and third complementarity-determining regions (CDR1, CDR2, and CDR3) having the amino acid sequences set forth in SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:32.
[0110] In other embodiments, the TCR has (i) an alpha chain having first, second and third complementarity-determining regions (CDR1, CDR2, and CDR3) having the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:34, respectively; and (ii) a beta chain having first, second and third complementarity-determining regions (CDR1, CDR2, and CDR3) having the amino acid sequences set forth in SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:32.
[0111] In other embodiments, the TCR has (i) an alpha chain having first, second and third complementarity-determining regions (CDR1, CDR2, and CDR3) having the amino acid sequences set forth in SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:30, respectively; and (ii) a beta chain having first, second and third complementarity-determining regions (CDR1, CDR2, and CDR3) having the amino acid sequences set forth in SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:36.
[0112] In other embodiments, the TCR has (i) an alpha chain having first, second and third complementarity-determining regions (CDR1, CDR2, and CDR3) having the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:34, respectively; and (ii) a beta chain having first, second and third complementarity-determining regions (CDR1, CDR2, and CDR3) having the amino acid sequences set forth in SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:36.
[0113] In some embodiments, the antigen targeting agent has first and second chains, which may be formed as a single polypeptide or as two separate polypeptides, each of the first and second chains having CDRs, the CDRs independently having any combination of the sequences of the CDRs set forth in Table 4.
[0114] In some embodiments, the engineered antigen targeting receptor has any one of the amino acid sequences set forth in SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44.
[0115] In some embodiments, the engineered antigen targeting receptor is transduced into the T-cell using a viral vector having the nucleotide sequence of the plasmid of any one of SEQ ID NOs:45, 46, 47 or 48.
[0116] In some embodiments, the alpha chain and the beta chain of the TCRs are interchangeable, i.e. can be expressed in any desired order from a suitable expression vector. The variable domains of the α chain (V.sub.α) and the β chain (V.sub.β) comprise any pairwise combination of the variable regions and/or the CDRs of the sequences of SEQ ID NOs: 38, 40, 42 and 44.
[0117] Suitable variations on such constructs can be made by those skilled in the art, for example the antigen-binding domains of a T-cell receptor can be inserted into a CAR construct in place of the typical scFv fragment together so that the single-chain antigen-binding domain interacts with the signaling domain of the CAR construct to cause the desired cytotoxic activity towards cancer cells.
[0118] In some embodiments, the antigen targeting agent is a chimeric antigen receptor (CAR). In such embodiments, the CAR is structured to provide a single-chain antigen binding domain equivalent to the TCR binding domain described above having the first and second chains (e.g. alpha and beta chains) of the TCR (each having three complementarity determining regions, which may be any of the complementarity determining regions described above for the TCR construct) joined together as a single polypeptide and linked together to a single hinge region, transmembrane domain and signalling domain, as well as a suitable co-stimulatory domain, (e.g. CD27, CD28, 4-1BB, ICOS, OX40, MYD88, IL1R1, CD70, or the like), as well as any other domains intended to enhance the characteristics of the CAR construct.
[0119] In some embodiments, the antigen targeting agent is a bispecific antibody, wherein the bispecific antibody has a first antigen-binding domain that binds to a factor such as CD3 that can be used to recruit T-cells and a second antigen-binding domain that binds to a KRAS.sup.G12X mutant peptide displayed by an HLA-A*02 molecule, including an HLA-A*02:01 molecule. In one example embodiment, the second domain of the bispecific antibody has as a single polypeptide the first and second chains (e.g. alpha and beta chains) of a TCR as described herein (each having three complementarity determining regions, which may be any of the complementarity determining regions described herein for the TCR construct) to provide the second antigen-binding domain.
[0120] Some embodiments of the present invention relate to nucleic acids, recombinant vectors, host cells, populations of cells and pharmaceutical compositions relating to, incorporating or encoding the TCRs, polypeptides and proteins described above.
Conduct of Immunotherapy Using Antigen Targeting Agents
[0121] In some embodiments, the antigen targeting agents described above, such as TCRs or CARs, are introduced into cytotoxic cells in any suitable manner, to provide a cytotoxic cell that specifically targets and kills cells expressing a form of KRAS that is mutated at position 12 as presented by HLA-A*02 molecules such as HLA-A*02:01 molecules. In some embodiments, the mutant KRAS is KRAS.sup.G12D, KRAS.sup.G12V or KRAS.sup.G12C.
[0122] Examples of cytotoxic cells that can be used in various embodiments include tumour infiltrating lymphocytes (TILs), including CD8.sup.+ T-cells, CD4.sup.+ T-cells, natural killer (NK) cells, and the like. Any cell that can be engineered to carry out cellular immunotherapy can be used in alternative embodiments.
[0123] The antigen targeting construct can be introduced into the cytotoxic cell using any suitable technique now known or later developed. In some embodiments, the antigen targeting construct is introduced into the cytotoxic cell using plasmid or RNA transfection, transduction by viral vectors, direct editing via programmable nucleases (e.g. CRISPR systems (clustered regularly interspaced short palindromic repeats), TALENs (transcription activator-like effector nucleases), zinc finger nucleases, and so on as known to those skilled in the art. In some embodiments, the antigen targeting construct is introduced into the cytotoxic cell by transduction with a suitable a vector, e.g. lentiviral or retroviral vectors, adenoviruses, adeno-associated virus (AAV), transposons, and the like. In some embodiments, the antigen targeting construct is introduced into the cytotoxic cell using a transposon system or electroporation.
[0124] In some embodiments, the desired antigen targeting receptor is used to generate engineered cytotoxic cells using autologous adoptive cell therapy. That is, the cytotoxic cells are harvested from a mammalian subject, genetically engineered to express the antigen targeting receptor, expanded ex vivo, and then the expanded cells are introduced back into the subject to treat the cancer associated with cells expressing the mutant form of KRAS having a missense mutation at position 12, e.g. KRAS.sup.G12D, KRAS.sup.G12V or KRAS.sup.G12C. In some embodiments, the mammalian subject is a human.
[0125] In some embodiments, the desired antigen targeting receptor is used to generate engineered cytotoxic cells using universal adoptive cell therapy using allogenic cells. In universal adoptive cell therapy, a bank of cells from an allogenic donor are genetically modified to express the desired antigen targeting receptor, such as a TCR or CAR as described herein. The modified allogenic cells are then introduced into a patient to treat a cancer associated with cells expressing a mutant form of KRAS, e.g. KRAS.sup.G12D, KRAS.sup.G12V or KRAS.sup.G12C. The patient can be a mammalian subject, e.g. a human.
[0126] In some embodiments, the desired antigen targeting receptor is introduced into a mammalian subject, e.g. a human, using systemic gene therapy. For example, a replication incompetent viral vector containing a nucleotide sequence for expressing the antigen targeting receptor is directly infused into a patient to directly transduce T-cells in situ to treat a cancer associated with cells expressing a mutant form of KRAS, e.g. KRAS.sup.G12D, KRAS.sup.G12V or KRAS.sup.G12C.
[0127] In some embodiments rather than engineering cytotoxic cells, the desired antigen targeting receptor is converted into a suitable soluble immunotherapy agent, for example a bi-specific antibody such as a bi-specific T-cell engager (BITE®), that can be directly administered to a mammalian subject. In such an embodiment, the portions of the first and second chains that form the antigen-binding region (each containing first, second and third CDRs) are combined together as a single polypeptide that targets tumour cells expressing mutant KRAS as displayed by HLA-A*02 molecules, including HLA-A*02:01 molecules, and are expressed as a fusion protein together with a second antigen binding domain, e.g. an scFv that binds to T-cells e.g. via the CD3 receptor. The resulting fusion protein is purified and administered to the subject in any suitable manner to direct cytotoxic T-cells to the tumour cells.
[0128] Methods of administration of the cellular immunotherapy agents and immunotherapy agents described herein are known in the art, and may include, for example, intravenous or subcutaneous injection.
[0129] In some embodiments, the likelihood that a mammalian subject will benefit from therapy using an antigen targeting agent described herein are conducted prior to commencing such therapy. A sample from the subject is evaluated to determine if the subject may have potentially cancerous cells that have a missense mutation at position 12 of KRAS. For example, a sample of a tumour from the patient may be subjected to DNA sequencing or appropriate analytical techniques to determine the presence of such a mutation. The mammalian subject is also subjected to HLA typing, to determine if the subject has an HLA-A*02 allele and/or which HLA-A allele the subject has. If the subject has both potentially cancerous cells that have a missense mutation at position 12 of KRAS and an HLA-A*02 allele, including in some embodiments an HLA-A*02:01 allele, then the subject is a potential candidate for immunotherapy using the antigen targeting agents described herein.
[0130] In one specific example embodiment, engineered TCRs as described herein are incorporated into CD8+ T cells. When the T-cell receptor recognizes and bind to KRAS.sup.G12D/V/C antigens presented by HLA-A*02 molecules (e.g. HLA*02:01 molecules) on tumour cells, the CD8+ T cells are activated and can bind to the tumour cells and initiate a cytotoxic response to kill the tumour cells, e.g. through lysis of the tumour cells, secretion of cytokines, and/or secretion of effector molecules (e.g. perforins and granzymes).
[0131] In one specific example embodiment, the T-cell receptors are synthesized and reconstituted in CD8+ T cells using lentiviral transduction. The lentiviral transduction uses a nucleotide vector encoding a receptor comprising an antigen binding domain capable of binding to KRAS.sup.G12D/V/C antigens presented by HLA-A*02 molecules (e.g. HLA-A*02:01 molecules). In some embodiments, the nucleotide vector includes nucleotides having a DNA sequence of any one of SEQ ID NOs:37, 39, 41 or 43.
[0132] In some embodiments, immune cells capable of binding to KRAS.sup.G12D/V/C antigens and initiating a cytotoxic response are made. They are made by first isolating the immune cells from a source of cells and genetically engineering the immune cells to express a receptor comprising an antigen binding domain capable of binding to KRAS.sup.G12D/V/C antigens as displayed at the cell surface by HLA-A*02 molecules. In some aspects, the genetic engineering can be carried out using a lentiviral vector. The engineered immune cells can be introduced into the body of a patient having an HLA-A*02 subtype and suffering from cancer or another disorder involving expression of KRAS.sup.G12D/V/C to treat the cancer or the disorder. In some embodiments, the patient has an HLA-A*02:01 subtype.
[0133] The engineered CD8+ T cells may be used to treat a patient with cancer and/or to screen for cancer. Focusing on an example illustrating the treatment aspect, because KRAS.sup.G12DN is a prevalent and mutation in patients suffering from pancreatic ductal adenocarcinoma (PDAC), the engineered CD8+ T cells may be particularly effective as an immunotherapeutic for such pancreatic cancers. Additionally, KRAS.sup.G12X is the most common cancer hotspot mutation and HLA-A*02:01 is a prevalent HLA allele, so a large patient population stands to benefit, and such benefit extends beyond PDAC to other cancer types with these common mutations such as lung and colorectal adenocarcinoma.
[0134] In some embodiments, the engineered immunotherapy receptors targeting KRAS.sup.G12X antigens are used in a patient having an HLA-A*02 subtype in a method for treating or preventing cancer. For example, the method may be chimeric antigen receptor (CAR) T-cell therapy or T-cell receptor (TCR) T-cell therapy.
[0135] In some embodiments, methods of identification of patients responsive to treatment by the present invention based on tumour KRAS mutation screening, HLA typing or other methods of patient screening are also provided.
Screening Using Antigen Targeting Agents
[0136] In some embodiments, the antigen targeting agents targeting KRAS.sup.G12X antigens displayed at the cell surface by HLA-A*02 molecules are used to detect the presence of tumour cells in a sample such as a patient biopsy. In some such embodiments, detection is made by conducting an assay to evaluate the ability of cytotoxic cells expressing the antigen targeting receptor to kill tumour cells in a tumour cell culture derived from the sample, or by evaluating the expression of molecules that indicate activation of cytotoxic cells, such as interferon-gamma, when such cells are co-cultured with tumour cells (e.g. using ELISpot).
[0137] In some embodiments, the antigen targeting agents targeting KRAS.sup.G12X antigens are used to detect the presence of tumour cells in a sample such as blood, for example by detecting such antigens displayed on episomes, i.e. membrane fragments that have been shown to be present in blood. In some embodiments, an in vitro assay using the synthetic TCRs, for example using the TCR as a labelled soluble reagent or expressed in a cell with a reporter system as described below can detect the presence of such antigens displayed on episomes.
[0138] In some embodiments, the engineered antigen targeting receptors are used for detecting the presence of cancer in a mammal. For example, the engineered antigen targeting receptors (their related polypeptides, proteins, nucleic acids, recombinant expression vectors, or engineered cells) may be brought into contact with a sample having one or more cells or episomes. If the cells express KRAS.sup.G12X antigens that are displayed by HLA-A*02 molecules, the engineered antigen targeting receptors will bind to the KRAS.sup.G12X antigens and thereby form a complex. The detection of the complex is indicative of the presence of potentially cancerous or pre-cancerous cells.
[0139] The detection of the complex may take place through any number of ways known in the art. In some embodiments, the engineered antigen targeting agents (and/or their related polypeptides, proteins, nucleic acids, recombinant expression vectors, or engineered cells) may be labeled with a detectable and/or visual label, e.g. a radioisotope or a fluorophore.
[0140] In some embodiments, the engineered antigen targeting receptors are reconstituted in immortalized T-cell lines (e.g. Jurkat cells) to support in vitro high throughput screening assays, for example for use in research and development and/or drug discovery. By way of non-limiting example, in some embodiments, the antigen targeting receptors are reconstituted in a soluble tetrameric form of an αβ TCR, i.e. a TCR multimer, and used diagnostically, e.g. to visualize cells exposed to infectious agents or cellular transformation and/or therapeutically, e.g. for the delivery of drugs to compromised cells, for example as described by Low et al. PloS One, 7(12), e51397, 2012. In some other embodiments, the engineered antigen targeting receptors are reconstituted in reporter cells derived from the T cell lymphoma line Jurkat as reported by Rydzek et al., Molecular Therapy, 27(2), 287-299, 2019.
EXAMPLES
[0141] Certain embodiments are further described with reference to the following examples, which are intended to be illustrative and not limiting in nature.
Example 1—Isolation of HLA-A*02:01:KRAS.SUP.G12D&V .Reactive CD8.SUP.+ T Cells
[0142] Clonally pure populations of HLA-A*02:01:KRAS.sup.G12D&V-reactive CD8.sup.+ T cells were isolated from peripheral blood mononuclear cells (PBMC) from a pancreatic cancer patient. Their target specificity to KRAS.sup.G12D&V antigens displayed by HLA-A*02:01 molecules was verified.
[0143] The TCR alpha and beta chains from HLA-A*02:01:KRAS.sup.G12D&V-reactive CD8.sup.+ T cell clones were sequenced, resynthesized and reconstituted as recombinant TCRs in healthy donor CD8+ T cells using lentiviral transduction.
[0144] The screening protocol to identify HLA-A*02:01:KRAS.sup.G12D&V-reactive CD8.sup.+ T cells was a modified “mini-line” culture method. The protocol is described in e.g. Wick et al., Clinical Cancer Research. 2014 Mar. 1; 20(5):1125-34. doi: 10.1158/1078-0432.CCR-13-2147. PMID: 24323902; Martin et al., A library-based screening method identifies neoantigen-reactive T cells in peripheral blood prior to relapse of ovarian cancer. Oncolmmunology. 2017 Sep. 21; 7(1):e1371895. doi: 10.1080/2162402X.2017.1371895. eCollection 2017. PMID: 29296522. Each of the foregoing publications is incorporated by reference herein.
[0145] The modified mini-line T-cell expansion protocol is schematically shown in
Example 2—Screening for Reactivity to KRAS.SUP.G12DN .Peptides
[0146] The panel of polyclonal T-cell pools was then screened for reactivity to KRAS.sup.G12DN peptides in the context of HLA-A*02:01 using IFN-γ (interferon gamma) ELISPOT assays (MabTech).
[0147] As shown in
[0148] As shown in
[0149] Single sorted T-cells were expanded in cRPMI media supplemented with IL-2 (200U/mL) and an excess of allogeneic irradiated PBMC feeders. To explore the function and specificity of anti-KRAS.sup.G12X monoclonal T-cell populations, some of the candidate T-cell clones were assessed by HLA-A*02:01-KRAS.sup.G12X tetramer staining (as shown in
[0150] With reference to
[0151] With reference to
[0152] Table 1 below summarizes the IFNγ ELISpot data as interpreted from the raw data, sample results of which are presented in
TABLE-US-00001 TABLE 1 Summary of example IFNγ ELISpot data. KCTL-2 (SFU of p value (two- IFNγ/2.5 × 10.sup.4 cell input) Mean SD N tailed T test) PANC-1 97 129 103 100 41 146 86 34.9 6 ** 0.0002 HeLa 8 6 11 2 0 0 4 4.7 6
[0153] The above data show cytolytic activity of the candidate TCRs is target specific. That is, there is selectivity towards the cognate neoantigen (G12D or G12V) used to isolate each TCR, and no specific recognition of the wild-type version of the KRAS 5-14aa epitope.
Example 3—Prediction for Binding of Different HLA-A*02 Subtypes to KRAS.SUP.G12D/V/C .Peptides
[0154] Binding predictions for various HLA-A*02 alleles to KRAS.sup.G12D/V/C peptides were carried out using NetMHCpan v3.0 (Nielsen, M., & Andreatta, M. (2016), Genome Medicine, 8(1), 33). An IC.sub.50 threshold of 500 nM was used to distinguish binding (IC.sub.50<500 nM) from non-binding peptides (IC.sub.50>500 nM). The HLA-A*02 alleles that are predicted to bind to KRAS.sup.G12D/V/C peptides are shown in Table 2.
[0155] About 154 distinct HLA-A*02 alleles were predicted to be able to bind to KRAS.sup.G12D. About 184 distinct HLA-A*02 alleles were predicted to be able to bind to KRAS.sup.G12V. About 180 distinct HLA-A*02 alleles were predicted to be able to bind to KRAS.sup.G12C.
TABLE-US-00002 TABLE 2 HLA-A*02 alleles predicted to bind to various KRAS.sup.G12X peptides and predicted binding affinity (IC.sub.50, nM). G12V G12C G12D Allele IC.sub.50 Allele IC.sub.50 Allele IC.sub.50 HLA-A02: 253 37.5 HLA-A02: 03 32.1 HLA-A02: 253 43.3 HLA-A02: 03 37.5 HLA-A02: 253 32.1 HLA-A02: 03 43.3 HLA-A02: 264 37.5 HLA-A02: 230 32.1 HLA-A02: 264 43.3 HLA-A02: 258 37.5 HLA-A02: 258 32.1 HLA-A02: 258 43.3 HLA-A02: 230 37.5 HLA-A02: 264 32.1 HLA-A02: 230 43.3 HLA-A02: 69 37.6 HLA-A02: 11 36.1 HLA-A02: 69 67.2 HLA-A02: 11 37.6 HLA-A02: 69 36.1 HLA-A02: 11 67.2 HLA-A02: 128 58.3 HLA-A02: 128 59.2 HLA-A02: 104 78 HLA-A02: 104 65.6 HLA-A02: 22 59.3 HLA-A02: 22 78 HLA-A02: 22 65.6 HLA-A02: 104 59.3 HLA-A02: 50 83.9 HLA-A02: 50 71.5 HLA-A02: 50 64 HLA-A02: 128 107.2 HLA-A02: 26 79 HLA-A02: 26 80.4 HLA-A02: 26 112.5 HLA-A02: 171 79 HLA-A02: 171 80.4 HLA-A02: 171 112.5 HLA-A02: 141 87.5 HLA-A02: 99 88.8 HLA-A02: 99 116.6 HLA-A02: 99 90.9 HLA-A02: 13 102.2 HLA-A02: 102 139.2 HLA-A02: 13 109.7 HLA-A02: 02 108.8 HLA-A02: 155 139.2 HLA-A02: 90 111.3 HLA-A02: 63 108.8 HLA-A02: 63 139.2 HLA-A02: 158 111.3 HLA-A02: 102 108.8 HLA-A02: 02 139.2 HLA-A02: 131 112.1 HLA-A02: 115 108.8 HLA-A02: 186 139.2 HLA-A02: 16 112.1 HLA-A02: 209 108.8 HLA-A02: 115 139.2 HLA-A02: 102 123.9 HLA-A02: 155 108.8 HLA-A02: 209 139.2 HLA-A02: 155 123.9 HLA-A02: 186 108.8 HLA-A02: 47 163 HLA-A02: 63 123.9 HLA-A02: 141 113.3 HLA-A02: 13 167.5 HLA-A02: 02 123.9 HLA-A02: 90 119.8 HLA-A02: 141 191.7 HLA-A02: 186 123.9 HLA-A02: 47 122.1 HLA-A02: 90 220.4 HLA-A02: 115 123.9 HLA-A02: 158 128.5 HLA-A02: 148 226.3 HLA-A02: 209 123.9 HLA-A02: 16 149.6 HLA-A02: 158 233.2 HLA-A02: 47 138.8 HLA-A02: 131 149.6 HLA-A02: 131 237.4 HLA-A02: 29 142.1 HLA-A02: 148 163.8 HLA-A02: 16 237.4 HLA-A02: 263 142.2 HLA-A02: 263 176.8 HLA-A02: 263 306.1 HLA-A02: 116 152.8 HLA-A02: 29 178.1 HLA-A02: 116 315.6 HLA-A02: 241 162.7 HLA-A02: 12 178.9 HLA-A02: 29 320.5 HLA-A02: 71 162.7 HLA-A02: 116 185.1 HLA-A02: 35 341.9 HLA-A02: 59 162.7 HLA-A02: 27 189.4 HLA-A02: 38 348.1 HLA-A02: 40 162.7 HLA-A02: 105 196.9 HLA-A02: 105 354 HLA-A02: 166 162.7 HLA-A02: 73 203.4 HLA-A02: 12 356.3 HLA-A02: 238 162.7 HLA-A02: 245 203.4 HLA-A02: 245 357.4 HLA-A02: 176 162.7 HLA-A02: 01 203.6 HLA-A02: 73 357.4 HLA-A02: 75 162.7 HLA-A02: 09 203.6 HLA-A02: 241 360.8 HLA-A02: 30 162.7 HLA-A02: 31 203.6 HLA-A02: 71 360.8 HLA-A02: 174 162.7 HLA-A02: 40 203.6 HLA-A02: 59 360.8 HLA-A02: 266 162.7 HLA-A02: 24 203.6 HLA-A02: 40 360.8 HLA-A02: 187 162.7 HLA-A02: 25 203.6 HLA-A02: 166 360.8 HLA-A02: 85 162.7 HLA-A02: 30 203.6 HLA-A02: 238 360.8 HLA-A02: 165 162.7 HLA-A02: 59 203.6 HLA-A02: 176 360.8 HLA-A02: 160 162.7 HLA-A02: 66 203.6 HLA-A02: 75 360.8 HLA-A02: 183 162.7 HLA-A02: 67 203.6 HLA-A02: 30 360.8 HLA-A02: 189 162.7 HLA-A02: 68 203.6 HLA-A02: 174 360.8 HLA-A02: 138 162.7 HLA-A02: 70 203.6 HLA-A02: 266 360.8 HLA-A02: 228 162.7 HLA-A02: 71 203.6 HLA-A02: 187 360.8 HLA-A02: 260 162.7 HLA-A02: 74 203.6 HLA-A02: 85 360.8 HLA-A02: 107 162.7 HLA-A02: 75 203.6 HLA-A02: 165 360.8 HLA-A02: 215 162.7 HLA-A02: 77 203.6 HLA-A02: 160 360.8 HLA-A02: 182 162.7 HLA-A02: 85 203.6 HLA-A02: 183 360.8 HLA-A02: 09 162.7 HLA-A02: 86 203.6 HLA-A02: 189 360.8 HLA-A02: 192 162.7 HLA-A02: 89 203.6 HLA-A02: 138 360.8 HLA-A02: 163 162.7 HLA-A02: 93 203.6 HLA-A02: 228 360.8 HLA-A02: 221 162.7 HLA-A02: 95 203.6 HLA-A02: 260 360.8 HLA-A02: 159 162.7 HLA-A02: 96 203.6 HLA-A02: 107 360.8 HLA-A02: 194 162.7 HLA-A02: 97 203.6 HLA-A02: 215 360.8 HLA-A02: 140 162.7 HLA-A02: 107 203.6 HLA-A02: 182 360.8 HLA-A02: 206 162.7 HLA-A02: 109 203.6 HLA-A02: 09 360.8 HLA-A02: 74 162.7 HLA-A02: 111 203.6 HLA-A02: 192 360.8 HLA-A02: 198 162.7 HLA-A02: 118 203.6 HLA-A02: 163 360.8 HLA-A02: 123 162.7 HLA-A02: 119 203.6 HLA-A02: 221 360.8 HLA-A02: 95 162.7 HLA-A02: 120 203.6 HLA-A02: 159 360.8 HLA-A02: 168 162.7 HLA-A02: 173 203.6 HLA-A02: 194 360.8 HLA-A02: 150 162.7 HLA-A02: 174 203.6 HLA-A02: 140 360.8 HLA-A02: 210 162.7 HLA-A02: 175 203.6 HLA-A02: 206 360.8 HLA-A02: 86 162.7 HLA-A02: 176 203.6 HLA-A02: 74 360.8 HLA-A02: 235 162.7 HLA-A02: 177 203.6 HLA-A02: 198 360.8 HLA-A02: 237 162.7 HLA-A02: 181 203.6 HLA-A02: 123 360.8 HLA-A02: 208 162.7 HLA-A02: 212 203.6 HLA-A02: 95 360.8 HLA-A02: 212 162.7 HLA-A02: 213 203.6 HLA-A02: 168 360.8 HLA-A02: 201 162.7 HLA-A02: 214 203.6 HLA-A02: 150 360.8 HLA-A02: 120 162.7 HLA-A02: 215 203.6 HLA-A02: 210 360.8 HLA-A02: 240 162.7 HLA-A02: 216 203.6 HLA-A02: 86 360.8 HLA-A02: 211 162.7 HLA-A02: 218 203.6 HLA-A02: 235 360.8 HLA-A02: 175 162.7 HLA-A02: 220 203.6 HLA-A02: 237 360.8 HLA-A02: 162 162.7 HLA-A02: 221 203.6 HLA-A02: 208 360.8 HLA-A02: 121 162.7 HLA-A02: 202 203.6 HLA-A02: 212 360.8 HLA-A02: 89 162.7 HLA-A02: 203 203.6 HLA-A02: 201 360.8 HLA-A02: 220 162.7 HLA-A02: 204 203.6 HLA-A02: 120 360.8 HLA-A02: 164 162.7 HLA-A02: 205 203.6 HLA-A02: 240 360.8 HLA-A02: 190 162.7 HLA-A02: 206 203.6 HLA-A02: 211 360.8 HLA-A02: 157 162.7 HLA-A02: 207 203.6 HLA-A02: 175 360.8 HLA-A02: 96 162.7 HLA-A02: 208 203.6 HLA-A02: 162 360.8 HLA-A02: 256 162.7 HLA-A02: 210 203.6 HLA-A02: 121 360.8 HLA-A02: 234 162.7 HLA-A02: 211 203.6 HLA-A02: 89 360.8 HLA-A02: 97 162.7 HLA-A02: 237 203.6 HLA-A02: 220 360.8 HLA-A02: 204 162.7 HLA-A02: 238 203.6 HLA-A02: 164 360.8 HLA-A02: 70 162.7 HLA-A02: 239 203.6 HLA-A02: 190 360.8 HLA-A02: 77 162.7 HLA-A02: 240 203.6 HLA-A02: 157 360.8 HLA-A02: 93 162.7 HLA-A02: 241 203.6 HLA-A02: 96 360.8 HLA-A02: 181 162.7 HLA-A02: 132 203.6 HLA-A02: 256 360.8 HLA-A02: 111 162.7 HLA-A02: 133 203.6 HLA-A02: 234 360.8 HLA-A02: 118 162.7 HLA-A02: 134 203.6 HLA-A02: 97 360.8 HLA-A02: 196 162.7 HLA-A02: 138 203.6 HLA-A02: 204 360.8 HLA-A02: 185 162.7 HLA-A02: 140 203.6 HLA-A02: 70 360.8 HLA-A02: 214 162.7 HLA-A02: 153 203.6 HLA-A02: 77 360.8 HLA-A02: 193 162.7 HLA-A02: 157 203.6 HLA-A02: 93 360.8 HLA-A02: 200 162.7 HLA-A02: 159 203.6 HLA-A02: 181 360.8 HLA-A02: 25 162.7 HLA-A02: 160 203.6 HLA-A02: 111 360.8 HLA-A02: 173 162.7 HLA-A02: 162 203.6 HLA-A02: 118 360.8 HLA-A02: 177 162.7 HLA-A02: 163 203.6 HLA-A02: 196 360.8 HLA-A02: 207 162.7 HLA-A02: 164 203.6 HLA-A02: 185 360.8 HLA-A02: 257 162.7 HLA-A02: 165 203.6 HLA-A02: 214 360.8 HLA-A02: 203 162.7 HLA-A02: 166 203.6 HLA-A02: 193 360.8 HLA-A02: 199 162.7 HLA-A02: 168 203.6 HLA-A02: 200 360.8 HLA-A02: 66 162.7 HLA-A02: 251 203.6 HLA-A02: 25 360.8 HLA-A02: 01 162.7 HLA-A02: 252 203.6 HLA-A02: 173 360.8 HLA-A02: 216 162.7 HLA-A02: 256 203.6 HLA-A02: 177 360.8 HLA-A02: 133 162.7 HLA-A02: 257 203.6 HLA-A02: 207 360.8 HLA-A02: 119 162.7 HLA-A02: 145 203.6 HLA-A02: 257 360.8 HLA-A02: 153 162.7 HLA-A02: 149 203.6 HLA-A02: 203 360.8 HLA-A02: 251 162.7 HLA-A02: 150 203.6 HLA-A02: 199 360.8 HLA-A02: 145 162.7 HLA-A02: 192 203.6 HLA-A02: 66 360.8 HLA-A02: 24 162.7 HLA-A02: 193 203.6 HLA-A02: 01 360.8 HLA-A02: 197 162.7 HLA-A02: 194 203.6 HLA-A02: 216 360.8 HLA-A02: 236 162.7 HLA-A02: 196 203.6 HLA-A02: 133 360.8 HLA-A02: 149 162.7 HLA-A02: 197 203.6 HLA-A02: 119 360.8 HLA-A02: 68 162.7 HLA-A02: 198 203.6 HLA-A02: 153 360.8 HLA-A02: 218 162.7 HLA-A02: 199 203.6 HLA-A02: 251 360.8 HLA-A02: 205 162.7 HLA-A02: 200 203.6 HLA-A02: 145 360.8 HLA-A02: 31 162.7 HLA-A02: 201 203.6 HLA-A02: 24 360.8 HLA-A02: 239 162.7 HLA-A02: 228 203.6 HLA-A02: 197 360.8 HLA-A02: 109 162.7 HLA-A02: 234 203.6 HLA-A02: 236 360.8 HLA-A02: 67 162.7 HLA-A02: 235 203.6 HLA-A02: 149 360.8 HLA-A02: 132 162.7 HLA-A02: 236 203.6 HLA-A02: 68 360.8 HLA-A02: 134 162.7 HLA-A02: 260 203.6 HLA-A02: 218 360.8 HLA-A02: 252 162.7 HLA-A02: 266 203.6 HLA-A02: 205 360.8 HLA-A02: 202 162.7 HLA-A02: 182 203.6 HLA-A02: 31 360.8 HLA-A02: 213 162.7 HLA-A02: 183 203.6 HLA-A02: 239 360.8 HLA-A02: 35 163.8 HLA-A02: 185 203.6 HLA-A02: 109 360.8 HLA-A02: 161 166.2 HLA-A02: 187 203.6 HLA-A02: 67 360.8 HLA-A02: 245 166.6 HLA-A02: 189 203.6 HLA-A02: 132 360.8 HLA-A02: 73 166.6 HLA-A02: 190 203.6 HLA-A02: 134 360.8 HLA-A02: 105 172.3 HLA-A02: 121 203.6 HLA-A02: 252 360.8 HLA-A02: 12 172.7 HLA-A02: 123 203.6 HLA-A02: 202 360.8 HLA-A02: 27 189.1 HLA-A02: 161 208.6 HLA-A02: 213 360.8 HLA-A02: 148 198.3 HLA-A02: 35 211 HLA-A02: 161 371 HLA-A02: 139 200.4 HLA-A02: 38 216.6 HLA-A02: 122 376.5 HLA-A02: 78 212.1 HLA-A02: 139 240 HLA-A02: 27 392.6 HLA-A02: 262 213.2 HLA-A02: 262 240.9 HLA-A02: 262 405 HLA-A02: 38 221.4 HLA-A02: 41 247.7 HLA-A02: 233 412.4 HLA-A02: 41 221.5 HLA-A02: 58 279.9 HLA-A02: 41 425.7 HLA-A02: 167 230.1 HLA-A02: 233 288.9 HLA-A02: 139 439.8 HLA-A02: 58 235.2 HLA-A02: 147 299.3 HLA-A02: 44 468.5 HLA-A02: 34 239.2 HLA-A02: 151 299.3 HLA-A02: 142 468.5 HLA-A02: 20 251.9 HLA-A02: 167 305.1 HLA-A02: 58 470.4 HLA-A02: 233 261.8 HLA-A02: 20 309.4 HLA-A02: 229 474.1 HLA-A02: 147 275.3 HLA-A02: 122 312.8 HLA-A02: 167 486 HLA-A02: 151 275.3 HLA-A02: 44 325.5 HLA-A02: 147 495.7 HLA-A02: 42 289.3 HLA-A02: 142 325.5 HLA-A02: 151 495.7 HLA-A02: 60 324.7 HLA-A02: 34 332.2 HLA-A02: 62 337.7 HLA-A02: 42 340.2 HLA-A02: 126 345.7 HLA-A02: 78 363.6 HLA-A02: 51 345.7 HLA-A02: 06 369.7 HLA-A02: 61 345.7 HLA-A02: 21 369.7 HLA-A02: 79 345.7 HLA-A02: 28 369.7 HLA-A02: 137 345.7 HLA-A02: 51 369.7 HLA-A02: 170 345.7 HLA-A02: 61 369.7 HLA-A02: 06 345.7 HLA-A02: 72 369.7 HLA-A02: 28 345.7 HLA-A02: 79 369.7 HLA-A02: 72 345.7 HLA-A02: 91 369.7 HLA-A02: 259 345.7 HLA-A02: 106 369.7 HLA-A02: 180 345.7 HLA-A02: 180 369.7 HLA-A02: 91 345.7 HLA-A02: 137 369.7 HLA-A02: 248 345.7 HLA-A02: 170 369.7 HLA-A02: 106 345.7 HLA-A02: 248 369.7 HLA-A02: 144 345.7 HLA-A02: 144 369.7 HLA-A02: 21 345.7 HLA-A02: 259 369.7 HLA-A02: 44 358.3 HLA-A02: 126 369.7 HLA-A02: 142 358.3 HLA-A02: 243 379.8 HLA-A02: 122 371.1 HLA-A02: 52 398.7 HLA-A02: 48 372 HLA-A02: 48 418.4 HLA-A02: 127 388.2 HLA-A02: 60 421.2 HLA-A02: 52 391.1 HLA-A02: 62 473.9 HLA-A02: 254 434.1 HLA-A02: 127 479.9 HLA-A02: 243 457.3 HLA-A02: 229 487.6 HLA-A02: 224 458.7 HLA-A02: 36 469 HLA-A02: 169 471.5 HLA-A02: 101 486.1
Example 4—Recombinant T-Cell Receptors
[0156] Candidate T-cell clones were then subjected to alpha-beta TCR amplification and sequencing. It was determined that KTCR-1 had the TRAV27*01 allele (SEQ ID NO:5 DNA and SEQ ID NO:6 amino acid) as the sequence for the variable region of the alpha chain of the TCR and the TRBV19*01 allele (SEQ ID NO:7 DNA and SEQ ID NO:8 amino acid) as the sequence for the beta chain of the TCR; that KTCR-2 had the TRAV13-2*01 allele (SEQ ID NO:9 DNA and SEQ ID NO:10 amino acid) as the sequence for the variable region of the alpha chain of the TCR and the TRBV19*01 allele (SEQ ID NO:7 DNA and SEQ ID NO:8 amino acid) as the sequence for the variable region of the beta chain of the TCR, and that KTCR-3 had the TRAV27*01 allele (SEQ ID NO:5 DNA and SEQ ID NO:6 amino acid) as the sequence for the variable region of the alpha chain of the TCR and the TRBV4-1*01 alelle (SEQ ID NO:11 DNA and SEQ ID NO:12 amino acid) as the sequence for the variable region of the beta chain of the TCR.
[0157] The alleles identified in the alpha and beta chains of the TCRs identified from KTCR-1, KTCR-2 and KTCR-3 are shown below in Table 3, along with the binding specificity of each (i.e. KRAS.sup.G12D or KRAS.sup.G12V). Based on these results, it is predicted that a TCR having the variable chain regions of TRAV13-2*01 for the alpha chain and TRBV04-1*01 for the beta chain of the TCR should also be effective in binding to KRASGI2X mutant peptides as presented by HLA-A*02:01. Such a construct is referred to herein as PTCR-4 as a predicted construct. Without being bound by theory, it is predicted that the PTCR-4 construct would recognize HLA-A*02:01 restricted KRAS.sup.G12D and KRAS.sup.G12V, but not KRAS.sup.Wild Type.
TABLE-US-00003 TABLE 3 Alleles for variable chain region of alpha and beta chains of sequenced TCRs. Alpha Chain Beta Chain Variable Region Variable Region TRBV 19*01 TRBV 04-1*01 TRAV27*01 KTCR-1 KTCR-3 (KRAS.sup.G12V) (KRAS.sup.G12D) TRAV13-2*01 KTCR-2 Predicted (KRAS.sup.G12D) (PTCR-4)
[0158] The variable region of each of the alpha and beta chains of the TCR containing the foregoing alleles contains the first and second complementarity determining region (CDR) of each chain (CDR1 and CDR2). The sequence of the third CDR was determined for each of KTCR-1, KTCR-2 and KTCR-3 to identify the sequences of each of the complementarity determining regions as follows in Table 4 and as underlined in
TABLE-US-00004 TABLE 4 Amino acid sequences of the first, second and third CDRs for each alpha and beta chain of each TCR. KTCR-1 KTCR-2 KTCR-3 (KRAS.sup.G12V) (KRAS.sup.G12D) (KRAS.sup.G12D) PTCR-4 CDR1-alpha SEQ ID NO: 14 SEQ ID NO: 18 SEQ ID NO: 14 SEQ ID NO: 18 CDR2-alpha SEQ ID NO: 16 SEQ ID NO: 20 SEQ ID NO: 16 SEQ ID NO: 20 CDR3-alpha SEQ ID NO: 30 SEQ ID NO: 34 SEQ ID NO: 30 SEQ ID NO: 34 CDR1-beta SEQ ID NO: 22 SEQ ID NO: 22 SEQ ID NO: 26 SEQ ID NO: 26 CDR2-beta SEQ ID NO: 24 SEQ ID NO: 24 SEQ ID NO: 28 SEQ ID NO: 28 CDR3-beta SEQ ID NO: 32 SEQ ID NO: 32 SEQ ID NO: 36 SEQ ID NO: 36
[0159] Recombinant TCRs for reconstitution were designed, incorporating the novel alpha-beta TCR sequences from the above three distinct T-cell clones, KTCR-1, KTCR-2 and KTCR-3, respectively. Physical DNA was synthesized de novo according to these designs, then ligated into lentiviral transfer plasmids shown schematically in
Example 5—Engineered CD8+ T Cells
[0160] Replication-incompetent lentiviral particles were then generated as TCR gene transfer vectors and used to transduce healthy donor CD8.sup.+ T-cells.
[0161]
[0162]
[0163] TCR-transduced CD8.sup.+ T cells were then evaluated for anti-KRAS.sup.G12X function and specificity by ELISPOT (as shown in
[0164]
[0165] Table 5 shows the results from ELISpot analysis of KTCR-1, KTCR-2, and KTCR-3 CD8.sup.+ T-cells. The results were reported as spot forming units (SFU) of gamma interferon (IFNγ). An ANOVA statistical analysis and a follow-up multiple comparison (Tukey's HSD multiple comparison test) were performed. A significant variance was found between KTCR-1 CD8.sup.+ T cells when co-cultured with HLA-A*02:01.sup.+ KRAS.sup.G12V CFPAC-1 cells, compared to the HLA-A*02:01.sup.− KRAS.sup.Wild type HeLa cells. Similarly, the KTCR-2, and KTCR-3 CD8.sup.+ T cells showed a significant increase in IFNγ SFUs when co-cultured with HLA-A*02:01.sup.+ KRAS.sup.G12D PANC-1 when compared to HLA-A*02:01.sup.+ KRAS.sup.G12V CFPAC-1 and HLA-A*02:01.sup.− KRAS.sup.Wild type HeLa cells. Data analysis was performed using Graphpad—Prism 8 (version 8.0.0).
TABLE-US-00005 TABLE 5 Analysis of KTCR-1, KTCR-2 and KTCR-3 CD8.sup.+ T-cells. SFU of IFNγ/ 2.0 × 10.sup.4 cell Multiple comparison input Mean SD N ANOVA test KTCR-1 PANC-1 13 18 19.5 9.19 2 P = 0.016 vs HLA-A*02:01.sup.+ KRAS.sup.G12D HeLa p = 0.818 CFPAC-1 52 42 42.0 14.14 2 vs Vs HLA-A*02:01.sup.+ KRAS.sup.G12V PANC-1 HeLa p = 0.025 p = 0.019 HeLa 2 13 7.5 7.78 2 HLA-A*02:01.sup.+ KRAS.sup.wild type KTCR-2 PANC-1 105 92 98.5 9.19 2 P < 0.001 vs vs HLA-A*02:01.sup.+ KRAS.sup.G12D CFPAC-1 HeLa p = 0.002 p = 0.002 CFPAC-1 8 15 11.5 4.95 2 vs HLA-A*02:01.sup.+ KRAS.sup.G12V HeLa p = 0.882 HeLa 11 14 12.5 2.12 2 HLA-A*02:01.sup.+ KRAS.sup.wild type KTCR-3 PANC-1 73 53 63.0 14.14 2 P = 0.016 vs vs HLA-A*02:01.sup.+ KRAS.sup.G12D CFPAC-1 HeLa p = 0.029 p = 0.018 CFPAC-1 13 19 21.0 2.83 2 vs HLA-A*02:01.sup.+ KRAS.sup.G12V HeLa p = 0.628 HeLa 7 6 6.5 0.71 2 HLA-A*02:01.sup.+ KRAS.sup.wild type
[0166]
[0167]
[0168]
[0169] Table 6 summarizes the data shown in
TABLE-US-00006 TABLE 6 Cell lysis of cells pulsed with KRAS.sup.G12X peptide and co-cultured with T-cells. Mean % SD N ANOVA Multiple comparison test K562_A*02:01 + KRAS.sup.G12D KTCR-1 4.0 2.47 4 P < 0.001 vs Control Lymphocytes p = 0.178 KTCR-2 19.1 2.99 4 vs KTCR-1 vs Control Lymphocytes vs KTCR-3 p < 0.001 p < 0.001 p = 0.503 KTCR-3 16.3 2.38 4 vs KTCR-1 vs Control Lymphocytes p < 0.001 p < 0.001 Control lymphocytes 0.1 0.02 4 K562_A*02:01 + KRAS.sup.G12V KTCR-1 16.5 2.41 4 p < 0.001 vs KTCR-2 vs KTCR-3 vs Control p < 0.001 p < 0.001 Lymphocytes p < 0.001 KTCR-2 4.0 2.61 4 vs Control Lymphocytes vs KTCR-3 p = 0.292 p = 0.678 KTCR-3 2.0 1.29 4 vs Control Lymphocytes p = 0.873 Control lymphocytes 0.6 0.97 4 K562_A*02:03 + KRAS.sup.Wild type KTCR-1 2.9 2.71 4 p = 0.272 vs KTCR-2 vs KTCR-3 vs Control p = 0.723 p > 0.999 Lymphocytes p = 0.419 KTCR-2 4.79 2.91 4 vs Control Lymphocytes vs KTCR-3 p = 0.075 p = 0.679 KTCR-3 2.82 4.08 4 vs Control Lymphocytes p = 0.459 Control lymphocytes 0.4 0.17 4
[0170] With reference to
[0171]
[0172]
[0173] The foregoing examples demonstrate that T-cells can be successfully transduced with engineered T-cell receptors that target KRAS.sup.G12X mutant peptides restricted and displayed by HLA-A*02:01, and that such T-cells can be used to kill cells that express the KRas having the relevant G12X mutation. Such cells have potential utility in the diagnosis, prophylaxis and/or treatment of cancers in which KRas that is mutated at position 12 is implicated in subjects having the HLA-A*02:01 allele. Based on computational analysis of the predicted binding of KRAS.sup.G12X mutant peptides as displayed by other HLA-A*02 alleles, it can be predicted that such cells have potential utility in the diagnosis, prophylaxis and/or treatment of cancers in which KRas that is mutated at position 12 is implicated in subjects having other HLA-A*02 alleles.
[0174] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are consistent with the broadest interpretation of the specification as a whole.
REFERENCES
[0175] The following references are of interest with respect to the subject matter described herein. The following references and all other references mentioned in this specification are incorporated by reference in their entireties. [0176] 1. Jones, S. et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321, 1801-6 (2008). [0177] 2. Weinstein, I. B. Cancer. Addiction to oncogenes—the Achilles heal of cancer.
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