Human alpha-fetoprotein specific T cell receptor and uses thereof
阅读说明：本技术 人甲胎蛋白特异性t细胞受体及其用途 (Human alpha-fetoprotein specific T cell receptor and uses thereof ) 是由 何玉凯 朱伟 E·塞利斯 彭一兵 王岚 于 2018-05-02 设计创作，主要内容包括：本发明提供了特异性识别hAFP<Sub>158</Sub>的T细胞受体及其使用方法。(The invention provides a method for specifically recognizing hAFP 158 And methods of using the same.)
1. An engineered mouse T cell receptor (mTCR) V.alpha.chain polypeptide having at least 90% sequence identity to SEQ ID NO 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein the mTCR specifically binds hAFP158(SEQ ID NO 1).
2. An engineered mouse T cell receptor (mTCR) V.beta.chain polypeptide having at least 90% sequence identity to SEQ ID NO 11, 12, 13, or 14, wherein the mTCR specifically binds hAFP158(SEQ ID NO: 1).
3. An engineered mouse T cell receptor (mTCR) full-length alpha chain polypeptide having at least 90% sequence identity to SEQ ID NO 15, 16, 17, 18, 19, 20, 21, 22, or 23, wherein the mTCR specifically binds hAFP158(SEQ ID NO: 1).
4. An engineered mouse T cell receptor (mTCR) full-length beta chain polypeptide having at least 90% sequence identity to SEQ ID NO:24, 25, 26, or 27, wherein the mTCR specifically binds hAFP158(SEQ ID NO: 1).
5. An engineered mouse T cell receptor (mTCR) alpha chain polypeptide comprising a CDR3 region having at least 90% sequence identity to SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35, or 36, wherein the mTCR specifically binds hAFP158(SEQ ID NO: 1).
6. An engineered mouse T cell receptor (mTCR) beta chain polypeptide comprising a CDR3 region having at least 90% sequence identity to SEQ ID NO:37, 38, 39, or 40, wherein the mTCR specifically binds hAFP158(SEQ ID NO: 1).
7. An engineered mouse T cell receptor (mTCR) comprising:
a) a V.alpha.domain having at least 90% sequence identity to SEQ ID NO. 2, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 28; and
b) a V.beta.domain having at least 90% sequence identity to SEQ ID NO. 11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 37.
8. An engineered mouse T cell receptor (mTCR) comprising:
a) a V.alpha.domain having at least 90% sequence identity to SEQ ID NO. 3, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 29; and
b) a V.beta.domain having at least 90% sequence identity to SEQ ID NO. 11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 37.
9. An engineered mouse T cell receptor (mTCR) comprising:
a) a V.alpha.domain having at least 90% sequence identity to SEQ ID NO. 4, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 30; and
b) a V.beta.domain having at least 90% sequence identity to SEQ ID NO. 12, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 38.
10. An engineered mouse T cell receptor (mTCR) comprising:
a) a V.alpha.domain having at least 90% sequence identity to SEQ ID NO. 5, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 31; and
b) a V.beta.domain having at least 90% sequence identity to SEQ ID NO. 13, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 39.
11. An engineered mouse T cell receptor (mTCR) comprising:
a) a V.alpha.domain having at least 90% sequence identity to SEQ ID NO. 6, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 32; and
b) a V.beta.domain having at least 90% sequence identity to SEQ ID NO. 11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 37.
12. An engineered mouse T cell receptor (mTCR) comprising:
a) a V.alpha.domain having at least 90% sequence identity to SEQ ID NO. 7, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 33; and
b) a V.beta.domain having at least 90% sequence identity to SEQ ID NO. 14, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 40.
13. An engineered mouse T cell receptor (mTCR) comprising:
a) a V.alpha.domain having at least 90% sequence identity to SEQ ID NO. 8, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 34; and
b) a V.beta.domain having at least 90% sequence identity to SEQ ID NO. 11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 37.
14. An engineered mouse T cell receptor (mTCR) comprising:
a) a V.alpha.domain having at least 90% sequence identity to SEQ ID NO. 9, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 35; and
b) a V.beta.domain having at least 90% sequence identity to SEQ ID NO. 11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 37.
15. An engineered mouse T cell receptor (mTCR) comprising:
a) a V.alpha.domain having at least 90% sequence identity to SEQ ID NO. 10, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 36; and
b) a V.beta.domain having at least 90% sequence identity to SEQ ID NO. 11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO. 37.
16. The engineered mouse T cell receptor (mTCR) according to any one of claims 7 to 15, wherein the mTCR is humanized.
17. A T cell engineered to express the mTCR of any one of claims 7 to 16.
18. The T cell of claim 17, wherein the T cell is a human T cell.
19. The T cell of claim 18, wherein the T cell is an autologous T cell.
20. A soluble mTCR α chain comprising Q21 through L242 of SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, or 23.
21. An extracellular domain of an engineered mTCR beta chain comprising SEQ ID NO 50, 51, 52, or 53 or E18 through Y276 of SEQ ID NO 24, 25, 26, or 27.
22. A fusion protein having at least 90% sequence identity to SEQ ID No. 54, 55, 56, 57, 58, 59, 60, 61 or 62.
23. A nucleic acid having at least 90% sequence identity to SEQ ID NO 63, 64, 65, 66, 67, 68, 69, 70 or 71.
24. A non-naturally occurring hAFP158 epitope-specific mouse T cell receptor comprising:
a) an alpha chain variable domain (V.alpha.) having at least 90% sequence identity to amino acid residues 21-132 of SEQ ID NO 2, 3, 4, 5, 6, 7, 8, 9 or 10; and
b) a beta-chain variable domain (V.beta.) having at least 90% sequence identity to amino acid residues 18-131 of SEQ ID NO 11, 12, 13 or 14.
25. A fusion protein comprising a first polypeptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9 or 10 linked to a second polypeptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs 11, 12, 13 or 14.
26. A fusion protein comprising a first polypeptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs 15, 16, 17, 18, 19, 20, 21, 22 or 23 linked to a second polypeptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs 24, 25, 26 or 27.
27. A T cell engineered to express mTCR encoded by SEQ ID NO 63, 64, 65, 66, 67, 68, 69, 70 or 71.
28. The T cell of claim 27, wherein the T cell is a human T cell.
29. A hybridoma comprising a CD8+ Tet158+ cell fused to a donor cell lacking a TCR a chain and a β chain.
30. The hybridoma of claim 29, wherein said hybridoma is responsive to hAFP + tumor cells.
31. The hybridoma of claim 29, wherein said hybridoma secretes IL-2.
32. A fusion protein comprising a first polypeptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked to a second polypeptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs 11, 12, 13, 14, 24, 25, 26, or 27, wherein the fusion protein is linked to a single chain anti-CD 3 antibody.
33. A method for treating a tumor in a subject in need thereof, the method comprising genetically engineering human T cells to express the mTCR of any one of claims 7 to 16, and administering the engineered T cells to the subject in an amount effective to reduce the tumor burden of the subject.
34. The method of claim 33, wherein the tumor is hepatocellular carcinoma.
35. The method of claim 33, wherein the T cell is an autologous T cell.
36. The polypeptide or protein of any one of claims 1-15 and 24-26, further comprising a detectable label.
37. A method for detecting hepatocellular carcinoma, the method comprising contacting the polypeptide or protein of claim 36 with a sample of cells, wherein specific binding of the polypeptide or protein to cells is indicative of hepatocellular carcinoma cells.
38. An antibody or antigen-binding fragment thereof that specifically binds to the polypeptide or protein of any one of claims 1-6 and 20-22.
Technical Field
The present invention relates generally to immunology, and specifically to T cell receptors and methods of their use in treating immune diseases, including cancer.
Background
Hepatocellular carcinoma (HCC) is the 5 th most common cancer worldwide, with about 80 million new cases per year. According to the american cancer society's data, the number of liver cancers (most of which are HCC) has doubled in the united states over the last decade, representing one of the rapidly growing malignancies primarily caused by an epidemic of obesity. The incidence of HCC may be high due to the large number of existing chronic HBV and HCV patients and epidemic obesity. Worse still, the lack of effective management makes HCC the 2 nd leading cause of cancer death in adult men. Therefore, the development of new therapies is urgently required. Adoptive transfer of tumor-specific T cells has great potential in controlling tumor growth without significant toxicity. Due to the great difficulty in isolating tumor-specific T cells from most solid tumors (except melanoma), genetic engineering of autologous T cells from patients with tumor antigen-specific TCR genes would likely provide functional tumor-specific T cells for adoptive cell transfer immunotherapy.
HCC frequently re-expresses human glypican 3 (hpgpc 3) and human alpha-fetoprotein (hAFP) as tumor-associated antigens (TAA). These antigens are not only used as diagnostic biomarkers, but also as targets for immunotherapy. Recently, the hpgc 3-specific human TCR gene was cloned and demonstrated anti-tumor efficacy in immunocompromised mice using a xenografted HCC model (Dargel et al, 2015). However, since TAA is often expressed at unequal levels throughout HCC, anti-hpgc 3 treatment may select hpgc 3 negative cells, leading to relapse. In theory, using a combination of TCRs directed against different epitopes and different tumor antigens can avoid or delay tumor immune escape. Four HLA-A2-restricted hAFP epitopes have been identified (Butterfield et al, 2001). Epitope hAFP158Is usually presented by HCC tumor cells, and hAFP is found in HCC patients158Specific immune cells (Butterfield et al, 2003), but weak anti-tumor effects (Butterfield et al, 2006), probably due to human hAFP158The affinity of specific T cells is lower. Thus, high affinity hAFPs were found158Specific TCRs can increase the anti-tumor efficacy of the targeted AFP antigen.
Two hAFPs have been reported158A specific TCR gene. A more recent reference to hAFP158Specific for epitopesThe patent for human TCR (CN 104087592A) showed limited antitumor effect (Sun et al, 2016). This weak anti-tumor effect is further demonstrated by another patent application (US 2016/0137715A1) by Adaptimune, in which hAFP158The epitope-specific wild-type human TCR did not produce any effector function when co-cultured with human HCC tumor cells. Thus, hAFP was formed by mutating the CDR regions of a wild-type human TCR to increase recognition of HCC tumor cells158Specific high affinity TCRs and were patented (US 2016/0137715a 1). However, no clinical data are available to date indicating that such TCR-modified human T cells (TCR-T) are indeed capable of producing anti-tumor effects in vivo. Furthermore, according to recent reports, adoptive transfer of high affinity TCR-T cells is associated with severe off-target toxicity (Cameron et al, 2013; Linette et al, 2013; Morgan et al, 2013), and therefore more TCRs are expected to be available to increase the chance of finding an optimal TCR with high anti-tumor efficacy and low toxicity. Thus, there is a real need to identify additional TAA-specific TCRs that can be used to engineer autologous T cells of patients for immunotherapy.
Disclosure of Invention
The invention provides a method for specifically recognizing hAFP158The T cell receptor of (1). One embodiment provides an engineered mouse T cell receptor (mTCR) V.alpha.chain polypeptide having at least 90% sequence identity to SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein mTCR specifically recognizes hAFP158(SEQ ID NO:1)。
Another embodiment provides an engineered mouse T cell receptor (mTCR) V.beta.chain polypeptide having at least 90% sequence identity to SEQ ID NO 11, 12, 13, or 14, wherein the mTCR specifically recognizes hAFP158(SEQ ID NO:1)。
Another embodiment provides an engineered mouse T cell receptor (mTCR) full-length alpha chain polypeptide having at least 90% sequence identity to SEQ ID NO 15, 16, 17, 18, 19, 20, 21, 22, or 23, wherein the mTCR specifically recognizes hAFP158(SEQ ID NO:1)。
Another embodiment provides an engineered mouse T cellFull-length beta chain polypeptide of the cellular receptor (mTCR) having at least 90% sequence identity with SEQ ID NO:24, 25, 26 or 27, wherein mTCR specifically recognizes hAFP158(SEQ ID NO:1)。
One embodiment provides an engineered mouse T cell receptor (mTCR) alpha chain polypeptide having a CDR3 region having at least 90% sequence identity to SEQ ID NO 28, 29, 30, 31, 32, 33, 34, 35, or 36, wherein the mTCR specifically recognizes an hAFP158(SEQ ID NO:1)。
Another embodiment provides an engineered mouse T cell receptor (mTCR) beta chain polypeptide having a CDR3 region having at least 90% sequence identity with SEQ ID NO 37, 38, 39, or 40, wherein the mTCR specifically recognizes the hAFP158(SEQ ID NO:1)。
The leader sequence of any of the disclosed polypeptide sequences may be removed.
One embodiment provides an engineered mouse T cell receptor (mTCR) having a va domain with at least 90% sequence identity to SEQ ID NO:2, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:28, and a ν β domain with at least 90% sequence identity to SEQ ID NO:11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO: 37.
Another embodiment provides an engineered mouse T cell receptor (mTCR) having a va domain with at least 90% sequence identity to SEQ ID NO:3, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:29, and a ν β domain with at least 90% sequence identity to SEQ ID NO:11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO: 37.
Another embodiment provides an engineered mouse T cell receptor (mTCR) having a V.alpha.domain having at least 90% sequence identity to SEQ ID NO:4, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:30, and a V.beta.domain having at least 90% sequence identity to SEQ ID NO:12, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO: 38.
Another embodiment provides an engineered mouse T cell receptor (mTCR) having a va domain with at least 90% sequence identity to SEQ ID No. 5, wherein the CDR3 region has at least 90% sequence identity to SEQ ID No. 31, and a ν β domain with at least 90% sequence identity to SEQ ID No. 13, wherein the CDR3 region has at least 90% sequence identity to SEQ ID No. 39.
One embodiment provides an engineered mouse T cell receptor (mTCR) having a va domain with at least 90% sequence identity to SEQ ID No. 6, wherein the CDR3 region has at least 90% sequence identity to SEQ ID No. 32, and a ν β domain with at least 90% sequence identity to SEQ ID No. 11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID No. 37.
Some embodiments provide an engineered mouse T cell receptor (mTCR) having a va domain with at least 90% sequence identity to SEQ ID NO:7, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:33, and a ν β domain with at least 90% sequence identity to SEQ ID NO:14, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO: 40.
Another embodiment provides an engineered mouse T cell receptor (mTCR) having a V.alpha.domain having at least 90% sequence identity to SEQ ID NO:8, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:34, and a V.beta.domain having at least 90% sequence identity to SEQ ID NO:11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO: 37.
Another embodiment provides an engineered mouse T cell receptor (mTCR) having a va domain with at least 90% sequence identity to SEQ ID NO:9, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:35, and a ν β domain with at least 90% sequence identity to SEQ ID NO:11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO: 37.
One embodiment provides an engineered mouse T cell receptor (mTCR) having a va domain with at least 90% sequence identity to SEQ ID NO:10, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:36, and a ν β domain with at least 90% sequence identity to SEQ ID NO:11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO: 37. Any of the disclosed mtcrs can be humanized.
One embodiment provides a T cell engineered to express any of the disclosed mtcrs. The T cell may be a human T cell. The T cells may also be autologous T cells.
Another embodiment provides a soluble mTCR α chain comprising Q21 through L242 of SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, or 23.
Another embodiment provides an extracellular domain of an engineered mTCR β chain comprising SEQ ID NOs 50, 51, 52, or 53 or E18 through Y276 of SEQ ID NOs 24, 25, 26, or 27.
One embodiment provides a fusion protein having at least 90% sequence identity to SEQ ID NO 54, 55, 56, 57, 58, 59, 60, 61 or 62.
Another embodiment provides a vector encoding either one of the mTCR proteins or polypeptides.
Another embodiment provides a nucleic acid having at least 90% sequence identity to SEQ ID NO 63, 64, 65, 66, 67, 68, 69, 70 or 71.
Another embodiment provides a non-naturally occurring hAFP158An epitope-specific mouse T cell receptor comprising:
a) an alpha chain variable domain (V.alpha.) having at least 90% sequence identity to amino acid residues 21-132 of SEQ ID NO 2, 3, 4, 5, 6, 7, 8, 9 or 10; and
b) having at least the amino acid residues 18 to 131 of SEQ ID NO 11, 12, 13 or 14
A beta chain variable domain (V.beta.) of 90% sequence identity.
Another embodiment provides a fusion protein comprising a first polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9 or 10 linked to a second polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID No. 11, 12, 13 or 14.
Another embodiment provides a fusion protein comprising a first polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID NO 15, 16, 17, 18, 19, 20, 21, 22 or 23 linked to a second polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID NO 24, 25, 26 or 27.
Another embodiment provides T cells engineered to express a TCR encoded by SEQ ID NOs 63, 64, 65, 66, 67, 68, 69, 70 or 71. The T cell may be a human T cell.
Another embodiment provides a hybridoma having CD8+ Tet fused to a donor cell lacking TCR alpha and beta chains158+A cell. Hybridomas typically respond to hAFP + tumor cells. In certain embodiments, the hybridoma secretes IL-2.
Another embodiment provides a fusion protein having a first polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9 or 10 linked to a second polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID No. 11, 12, 13, 14, 24, 25, 26 or 27, wherein the fusion protein is linked to a single chain anti-CD 3 antibody.
Another embodiment provides a method for treating a tumor in a subject in need thereof: the engineered T cells are administered to a subject by genetically engineering human T cells to express the disclosed mtcrs in an amount effective to reduce the tumor burden of the subject. In certain embodiments, the tumor is hepatocellular carcinoma. The T cells may be autologous T cells.
Another embodiment provides a method for detecting hepatocellular carcinoma: by contacting the disclosed polypeptide or protein with a sample of cells, wherein specific binding of the polypeptide or protein to the cells is indicative of hepatocellular carcinoma cells.
Another embodiment provides an antibody or antigen-binding fragment thereof that specifically binds to the disclosed polypeptide or protein.
Drawings
FIGS. 1A-1 through 1A-4 are dot plots of CD8 and IFN-. gamma.in mouse peripheral blood cells stimulated with hAFP158 peptide by gating on Thy1.2+ T cells. FIGS. 1A-5 are graphs of% CD8+ IFNg + in total splenocytes with and without lv priming. FIGS. 1B-1 through 1B-3 are dot plots of splenic T cells stimulated with HepG2(AFP-) (FIG. 1B-1), HepG2(AFP +) (FIG. 1B-2), and hAFP158 peptide (FIG. 1B-3). FIGS. 1B-4 are graphs of% CD8+ IFNg + in total splenocytes stimulated with HepG2(AFP-), HepG2(AFP +) and hAFP158 peptides. FIGS. 1C-1 to 1C-3 are micrographs of HepG2 cells co-cultured with spleen cells. FIGS. 1C-4 are graphs of remaining viable HepGe cells (OD595) versus E/T ratio, (●) Lv + Pep, (■) Pep + Pep.
Fig. 2A to 2E show that adoptive transfer of splenocytes from immunized AAD mice protects mice from tumor challenge and eradicates HepG2 tumor xenografts in NSG mice. Fig. 2A is a dot plot showing that about 10% of immunized mouse spleen cells reacted to hAFP158 peptide to generate IFNg. Figure 2B shows a total of 1500 ten thousand splenocytes (150 ten thousand hAFP 158-specific CD 8T cells) from an unimmunized or immunized mouse were injected into NSG mice and then challenged with HepG2 tumor cells. FIG. 2C is a graph showing that 150 ten thousand spleen cells of an immunized mouse were injected into an NSG mouse when the tumor size reached 2cm in diameter. Fig. 2D is a line graph of tumor size (mm3) versus days post tumor inoculation in control (●) and ACT (■). FIG. 2E is a line graph of tumor volume (mm3) in mouse 1(□), mouse 2(□), mouse 3(□), and mouse 4(□) versus days post tumor inoculation,
FIG. 3A is a dot plot showing the purity of hAFP 158-specific CD 8T cells. Figure 3B is a dot plot showing the percentage of cells producing hAFP 158-specific IFN γ. Fig. 3C is a dot plot showing the purity of Flu M1-specific CD 8T cells. Figure 3D is a dot plot showing the percentage of Flu M1-specific IFN γ -producing cells. FIG. 3E is a line graph of tumor volume (mm3) versus days post tumor inoculation for mice treated with CD8(□) for Lv + hAFP158 or CD8 for Flu + M1. Fig. 3F to 3H are dot plots of purified CD 8T cells from hAFP-immunized mice that were further separated into Tet158+ cells and Tet 158-cells by Tet158 tetramer staining and cell sorter. The purity before and after sorting is presented. FIG. 3I is a line graph of tumor volume (mm3) versus days post tumor inoculation for mice treated with Tet158+ (□) cells or Tet158- (□) cells. Fig. 3J to 3O are images of the mouse from fig. 3I.
FIG. 4A is a bar graph of IL-2(pg/ml) from T cell hybridoma clones. BW-Lyt2.4 fusion partner cells and 5 different hybridoma clones were co-cultured in triplicate with hAFP + (□) or hAFP- (□) HepG2 tumor cells and assayed for IL-2 production by ELISA. Fig. 4B to 4G are histograms of hybridoma clones stained with anti-V β 8.3 antibody. Fig. 4H to 4M are histograms of hybridoma clones stained with Tet158 tetramer.
FIG. 5A is a schematic representation of recombinant lv expressing the TCR gene. Pairs of TCR α and β chain genes were expressed as a single molecule under the control of the EF1 α promoter. A P2A sequence was inserted between them to allow for the generation of an equal number of TCR alpha and beta chains. FIGS. 5B to 5E are histograms of Tet158 tetramer staining of human T cell line Jurkat cells after transduction with three different TCR-lv. Histograms and MFIs presented. Fig. 5G to 5J, 5L to 5O and 5Q to 5T are dot plots of primary human T cells from 3 different donors transduced with TCR-lv showing the percentage of Tet158+ CD 8T cells and CD4T cells and the MFI. Mock transduced cells underwent the same treatment with CD3/CD28 without lv transduction or with GFP-lv transduction. Fig. 5F, 5K, 5P and 5U are bar graphs showing the MFI of Tet158 on transduced T cells from three different donors. TCR-T cells are shown. Only CD 8T cells or CD4T cells were gated and are shown in representative figures.
FIG. 6A is a bar graph of IFN-g (pg/ml) from primary human T cells transduced with the TCR gene and treated with AFP-HepG2 (shaded in grey), Huh7 (shaded in solid) or AFP + HepG2 (unshaded). FIGS. 6B to 6E are dot plots showing intracellular staining of IFN γ and IL-2 by CD 8. FIGS. 6F to 6I are dot plots showing intracellular staining of CD4 for IFN γ and IL-2. FIGS. 6J through 6O are dot plots and histograms showing the induction of CD8 and CD4TCR-T cell proliferation by hAFP + HepG2 tumor cells. The experiment was repeated twice with similar data.
FIG. 7A is a bar graph showing the percent killing of HLA-A2+ human primary T cells transduced with the TCR gene and treated with AFP-HepG2 (shaded in grey), Huh7 (shaded in solid) or AFP + HepG2 (unshaded). FIG. 7B is a line graph of percent killing versus E: T ratio for TCR (□) and mock (□). Fig. 7C, 7D, and 7E are dot plots showing donor CD8 TCR-T cells and CD4TCR-T cells isolated by magnetic beads after TCR gene transduction. FIGS. 7F to 7K are micrographs of mock transduced, CD4TCR-T or CD8 TCR-T co-cultures with HepG2 tumor cells. Photographs were taken after 2 hours and 24 hours of co-cultivation. Fig. 7E is a bar graph showing the results of LDH assays used to measure the percent killing of CD4 cells (black bars), CD8 cells (white bars), and total T cells (gray bars) in TCR or mock-transduced human T cells. Fig. 7M to 7O are photomicrographs of representative cells from fig. L.
FIG. 8 is a map of the lentiviral vector pCDH-EF 1-cotR-1 expressing TCR-1.
FIG. 9 is a map of the lentiviral vector pCDH-EF 1-cotR-2 expressing TCR-1.
FIG. 10 is a map of the lentiviral vector pCDH-EF 1-cotR-3 expressing TCR-1.
Fig. 11A is a dot plot showing the percentage of Tet158+ CD8+ splenocytes from immunized mice. Fig. 11B to 11P are histograms showing V β expression in CD8+, Tet158+ cells using different anti-V β chain antibodies.
Fig. 12A to 12D show transduction of primary human T cells with recombinant lv. FIG. 12A is a schematic representation of the GFP-lv promoter. Fig. 12B and 12C are representative images of primary T cells expressing GFP. Fig. 12D is a histogram of GFP + T cells.
FIG. 13A is a histogram showing the percentage of TCR-T cells stained with anti-V.beta.chain antibody. FIG. 13B is a histogram showing the percentage of TCR-T cells stained with Tet158 tetramer.
FIGS. 14A-14I show that human TCR-T cells specifically recognize HLA-A2 mouse spleen cells impacted by AFP158 peptide. FIG. 14A is a bar graph showing IFN-g production (pg/ml) in mock T cells, TCR 1T cells, TCR 2T cells or TCR 3T cells stimulated with AAD mouse spleen cells and not challenged with peptide (gray bars), challenged with influenza M1 peptide (black bars) or challenged with AFP158 peptide (white bars). Fig. 14B-14E are histograms showing the percentage of IFNg-stained human T cells (containing both CD4 and CD 8) stimulated with HLA-a2 cells pulsed with hAFP158 peptide. FIGS. 14F-14I are histograms showing the percentage of IL-2 stained human T cells (containing both CD4 and CD 8) stimulated with HLA-A2 cells pulsed with hAFP158 peptide.
FIGS. 15A-15T show that adoptive transfer of human TCR-T produces an anti-tumor effect against HepG2 tumor in NSG mice. FIG. 15A is a dot plot showing% Tet158+ cells in total human TCR-T cells. FIG. 15B is a line graph showing tumor growth (% of tumor-free mice) versus days post tumor inoculation in control and TCR-T groups. FIG. 15C is a line graph showing tumor size (mm3) versus days post tumor inoculation in control (●) and TCR-T (■). FIGS. 15D to 15K are photographs of HepG2 tumors at day 31 after inoculation in mock T-treated (D1-4) and TCR-T treated (D5-8) mice. Fig. 15L to 15O are representative dot plots showing the percentage of human CD45+ cells in total mouse blood cells. FIG. 15P is a line graph showing the percentage of human CD45+ cells in total mouse blood cells of control (●) and TCR-T (■). Fig. 15Q to 15R are representative dot plots showing the percentage of Tet158+ cells to transferred human T cells in NSG mice. Fig. 15S is a line graph showing the percentage of Tet158+ cells to total transferred human T cells versus post-ACT days. Fig. 15T is a line graph showing the percentage of Tet158+ cells to total transferred CD8(□) T cells and CD4(□) T cells as a function of post-ACT days.
FIG. 16 is a schematic comparison of the V regions of 9 mTCR sequences.
FIG. 17 is a schematic comparison of the amino acid sequences of the CDRs 3 of the 9 mTCR α chains (FIG. 17A) and β chains (FIG. 17B).
Detailed Description
I. Definition of
As used herein, the phrase "having antigen specificity" refers to a TCR that can specifically bind to and immunologically recognize a cancer antigen such that binding of the TCR to the cancer antigen elicits an immune response.
The term "Tet158"refers to HLA-A2/hAFP158A tetramer.
The term "hAFP158"refers to a human alpha-fetoprotein polypeptide having the amino acid sequence of FMNKFIYEI (SEQ ID NO: 1).
The abbreviation "TCR" refers to a T cell receptor, which is a specific receptor on the surface of T cells responsible for recognizing antigens presented by the Major Histocompatibility Complex (MHC).
As used herein, the term "antibody" is intended to mean an immunoglobulin molecule having an antigen recognition site that is a "variable region". The term "variable region" is intended to distinguish this domain of an immunoglobulin from a domain that is widely shared by antibodies, such as an antibody Fc domain. The variable regions include the "hypervariable regions" whose residues are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs" (i.e., typically about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and about residues 27-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al, "Sequences of Proteins of immunological Interest", 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. 1991) and/or those residues from the "hypervariable loop" (i.e., residues 26-32 (L1), residues 50-52 (L2) and residues 91-96 (L3) in the light chain variable domain and residues 26-32 (H1) in the heavy chain variable domain, Residues 53-55 (H2) and residues 96-101 (H3); chothia and Lesk, 1987, J.mol.biol. vol. 196, pp 901-917). "framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues defined herein. The term antibody includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodiesAntibodies, chimeric Antibodies, camelized Antibodies (see, e.g., Muydermans et al, 2001, Trends biochem. Sci. Vol.26, p.230; Nuttall et al, 2000, Cur. pharm. Biotech. Vol.1, p.253; Reichmann and Muydermans, 1999, J.Immunol. meth. Vol.231, p.25; International publications WO 94/04678 and WO 94/25591; U.S. Pat. No. 6,005,079), single chain fv (scFv) (see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, p.269-315, year), single chain Antibodies, disulfide-linked (sdFvs), intracellular Antibodies and anti-idiotypic Antibodies (including, e.g., anti-Fvs), including anti-Antibodies such as anti-Id). In particular, such antibodies include any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG)1、IgG2、IgG3、IgG4、IgA1And IgA2) Or a subclass of immunoglobulin molecules.
As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that comprise the complementarity determining regions ("CDRs") of the antibody and, optionally, the framework residues of the "variable region" antigen recognition site of the antibody, and that exhibit the ability to immunospecifically bind an antigen. Such fragments include Fab ', F (ab')2Fv, single chain (ScFv) and mutants thereof, naturally occurring variants and fusion proteins comprising the "variable region" antigen recognition site of an antibody, as well as heterologous proteins (e.g., toxins, antigen recognition sites for different antigens, enzymes, receptors or receptor ligands, etc.).
The term "fragment" as used herein refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues.
As used herein, the term "modulate" relates to the ability to alter an effect, outcome, or activity (e.g., signal transduction). Such modulation may be agonistic or antagonistic. Antagonism modulation may be partial (i.e., reduced but not abolished), or may abolish such activity completely (e.g., neutralization). Modulation may include internalization of the receptor upon antibody binding or reduction of receptor expression on the target cell. Agonistic modulation may enhance or otherwise increase or enhance activity (e.g., signal transduction). In yet another embodiment, such modulation may alter the nature of the interaction between the ligand and its cognate receptor, thereby altering the nature of the signal transduction elicited. For example, a molecule may alter the ability of such a molecule to bind to other ligands or receptors by binding to the ligand or receptor, thereby altering its overall activity. Preferably, such modulation will provide a measurable change in the activity of the immune system of at least 10%, more preferably a change in such activity of at least 50%, or a change in such activity of at least 2-fold, 5-fold, 10-fold, or still more preferably at least 100-fold.
The term "substantially" as used in the context of binding or exhibiting an effect is intended to mean that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule can substantially block the activity of a ligand or receptor if the degree of blocking is physiologically or therapeutically relevant (e.g., if the degree of blocking is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 97% as compared to complete blocking). Similarly, an immune specificity and/or characteristic is considered substantially the same if the degree of similarity in such specificity and characteristic is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 97% between one molecule and another.
As used herein, a "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such as an antibody having variable regions derived from a non-human antibody and human immunoglobulin constant regions.
As used herein, the term "humanized antibody" refers to an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (typically mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor" and the human immunoglobulin providing the framework is referred to as the "acceptor". Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85% -99%, preferably about 95% or more identical. Thus, all parts of the humanized immunoglobulin are substantially identical to the corresponding parts of the natural human immunoglobulin sequence, except for possible CDRs. Humanized antibodies are antibodies that include humanized light chains and humanized heavy chain immunoglobulins. For example, a humanized antibody will not encompass a typical chimeric antibody because, for example, the entire variable region of the chimeric antibody is non-human.
As used herein, the term "endogenous concentration" refers to the level at which the molecule is naturally expressed (i.e., in the absence of an expression vector or recombinant promoter) by the cell (which may be a normal cell, a cancer cell, or an infected cell).
As used herein, the terms "treating", "treating" and "therapeutic use" refer to the elimination, alleviation or amelioration of one or more symptoms of a disease or disorder.
As used herein, "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to mediate clinically relevant elimination, reduction, or amelioration of such symptoms. An effect is clinically relevant if the magnitude of the effect is sufficient to affect the health or prognosis of the receiving subject. A therapeutically effective amount may refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of disease (e.g., delay or minimize the spread of cancer). A therapeutically effective amount may also refer to the amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.
As used herein, the term "prophylactic agent" refers to an agent that can be used to prevent a disorder or disease before any symptoms of such disorder or disease are detected. A "prophylactically effective" amount is an amount of prophylactic agent sufficient to mediate such protection. A prophylactically effective amount may also refer to the amount of prophylactic agent that provides a prophylactic benefit in the prevention of disease.
As used herein, the term "cancer" refers to a tumor caused by abnormal uncontrolled growth of cells. As used herein, cancer specifically includes leukemia and lymphoma. The term "cancer" refers to a disease involving cells that may metastasize to a distal site and exhibit a phenotypic trait different from non-cancerous cells (e.g., colony formation in a three-dimensional substrate such as soft agar or formation of a tubular network or reticulated matrix in a three-dimensional basement membrane or extracellular matrix preparation). Non-cancer cells do not form colonies in soft agar and form distinct globular structures in a three-dimensional basement membrane or extracellular matrix preparation.
As used herein, "immune cell" refers to any cell from hematopoietic origin, including but not limited to T cells, B cells, monocytes, dendritic cells, and macrophages.
As used herein, "valency" refers to the number of binding sites available per molecule.
As used herein, the term "immunological", "immunological" or "immune" response is the receipt of a beneficial humoral (antibody-mediated) and/or cellular (mediated by antigen-specific T cells or their secretory products) response to a peptide in a patient. Such responses may be active responses induced by administration of the immunogen, or passive responses induced by administration of antibodies or primed T cells. Presentation of polypeptide epitopes in association with class I or class II MHC molecules activates antigen-specific CD4+T helper cell and/or CD8+Cytotoxic T cells, thereby eliciting a cellular immune response. This response may also involve the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, the activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immune response can be determined by proliferation (CD 4)+T cells) or CTL (cytotoxic T lymphocytes) assays. The relative contribution of humoral and cellular responses to the protective or therapeutic effects of an immunogen may be determined by immunizing a syngeneic animal with the immunogenAntibodies and T cells are isolated and measured for protective or therapeutic effects in a second subject.
As used herein, an "immunogenic agent" or "immunogen" is capable of inducing an immune response against itself when administered to a mammal (optionally in combination with an adjuvant).
As used herein, the terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans, rodents (such as mice and rats), and other experimental animals.
As used herein, the term "polypeptide" refers to a chain of amino acids of any length, regardless of whether it is modified (e.g., phosphorylated or glycosylated). The term polypeptide includes proteins and fragments thereof. Polypeptides may be "exogenous," meaning that they are "heterologous," i.e., foreign to the host cell utilized, such as a human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as sequences of amino acid residues. These sequences are written from left to right in the direction from the amino to the carboxy terminus. According to standard nomenclature, amino acid residue sequences are designated by either three-letter or one-letter codes as follows: alanine (Ala, a), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains the necessary properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Typically, the differences are limited such that the sequences of the reference polypeptide and the variant are very similar overall and identical in many regions. The variant and reference polypeptides may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions and/or deletions). The substituted or inserted amino acid residue may or may not be an amino acid residue encoded by the genetic code. Variants of a polypeptide may be naturally occurring, such as allelic variants, or may be variants that are not known to occur naturally.
Modifications and changes can be made to the structure of the polypeptides of the disclosure and still obtain molecules having similar characteristics to the polypeptides (e.g., conservative amino acid substitutions). For example, certain amino acids may be substituted for other amino acids in the sequence without significant loss of activity. Because the interactive capacity and nature of a polypeptide determines the biological functional activity of the polypeptide, certain amino acid sequence substitutions may be made in the polypeptide sequence, but still obtain a polypeptide with similar properties.
In making such changes, the hydropathic index of the amino acid may be considered. The importance of the hydrophilic amino acid index in conferring interactive biological functions on polypeptides is generally understood in the art. It is known that certain amino acids may be substituted for other amino acids having similar hydropathic indices or scores and still result in polypeptides having similar biological activities. Each amino acid has been assigned a hydrophilicity index based on its hydrophobicity and charge characteristics. These indices are: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).
It is believed that the relative hydrophilicity of the amino acids determines the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted for another amino acid with a similar hydropathic index and still result in a functionally equivalent polypeptide. In such variations, substitutions of amino acids with a hydrophilicity index within ± 2 are preferred, those with a hydrophilicity index within ± 1 are particularly preferred, and those with a hydrophilicity index within ± 0.5 are even more particularly preferred.
Substitutions of like amino acids may also be made on the basis of hydrophilicity, particularly where the resulting biologically functional equivalent polypeptide or peptide is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartic acid (+3.0 + -1), glutamic acid (+3.0 + -1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), proline (-0.5 + -1), threonine (-0.4), alanine (-0.5), histidine (-0.5), cysteine (-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5), tryptophan (-3.4). It is understood that one amino acid may be substituted for another amino acid having a similar hydrophilicity value and still result in a biologically equivalent, particularly immunologically equivalent polypeptide. In such variations, substitutions of amino acids having a hydrophilicity value within ± 2 are preferred, those having a hydrophilicity value within ± 1 are particularly preferred, and those having a hydrophilicity value within ± 0.5 are even more particularly preferred.
As noted above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account the various aforementioned characteristics are well known to those skilled in the art and include (original residues: exemplary substitutions): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Trp: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Accordingly, embodiments of the present disclosure contemplate functional or biological equivalents of the polypeptides described above. In particular, embodiments of the polypeptide may include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide of interest.
The term "percent (%) sequence identity" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in a reference nucleic acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent sequence identity can be accomplished in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN-2, or megalign (dnastar) software. Suitable parameters for measuring alignment can be determined by known methods, including any algorithm required to achieve maximum alignment over the full length of the sequences being compared.
For purposes herein, the calculation of% sequence identity (which may alternatively be expressed as a percentage of sequence identity that a given sequence C has or includes with respect to, with, or against a given nucleic acid sequence D) for a given nucleotide or amino acid sequence C with respect to, with, or against a given nucleic acid sequence D is as follows:
100 times the fraction W/Z,
wherein W is the number of nucleotides or amino acids scored as identical matches in the alignment of C and D of the program by the sequence alignment program, and wherein Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the% sequence identity of C with respect to D will not be equal to the% sequence identity of D with respect to C.
As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline solutions, water and emulsions (such as oil/water or water/oil emulsions), as well as various types of wetting agents.
As used herein, the terms "antigenic determinant" and "epitope" are used interchangeably and refer to a structure that is recognized by an antibody.
As used herein, a "conformational epitope" is an epitope that includes a discontinuous portion of the amino acid sequence of an antigen. Antibodies bind conformational epitopes based on the 3-dimensional surface characteristics, shape or tertiary structure of the antigen.
As used herein, a "linear epitope" is an epitope formed by a contiguous sequence of amino acids from an antigen. Linear epitopes typically comprise from about 5 to about 10 contiguous amino acid residues. Antibodies bind linear epitopes based on the primary sequence of the antigen.
As used herein, a "paratope," also referred to as an "antigen binding site," is the portion of an antibody that recognizes and binds an antigen.
As used herein, "adoptive cell transfer" or ACT is an immunotherapy in which the patient's own T cells are collected, expanded ex vivo, and then re-injected into the patient. Two types of ACT are Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR) T cell therapy. Both techniques improve the ability of T cell receptors to recognize and attack specific antigens. In CAR T cell therapy, T cells are engineered to produce receptors on their surface called chimeric antigen receptors. The receptor allows T cells to recognize and attach to antigens on tumor cells. In TCR-T cell therapy, T cells are collected from a patient, modified to express TCRs specific for tumor antigens, expanded ex vivo, and then re-injected into the patient.
T cell receptor composition
T cell receptor genes encoding hAFPs, preferably hAFPs expressed on the surface of cancer cells, are provided158) A receptor that binds specifically. Identification and synthesis of peptides specific for HLA-A2/hAFP158Nine pairs of mouse TCR alpha chain genes and beta chain genes. The amino acid and nucleic acid sequences of the TCR genes are provided below. Vectors comprising one or more TCR genes are also provided.
Another embodiment provides a hAFP158Specificity (Tet)158) Mouse CD 8T cells that recognize and kill human HepG2 cells in vitro and eradicate large HepG2 tumor xenografts in NSG mice.
Another embodiment provides a method of making a tablet made from Tet158T cell hybridoma made from CD 8T cells. T cell hybridomas can identify pairs of TCR alpha chain genes and beta chain genes. One embodiment provides a method for the treatment of HLA-A2/hAFP158Mouse TCR alpha chain gene and beta chain gene transduced healthy donor CD 8T cells. The genetically engineered TCR enables donor cells to recognize and effectively kill HepG2 tumor cells at very low E/T ratios. These specificitiesOn HLA-A2/hAFP158It is possible to modify and redirect autologous T cells of patients via adoptive cell transfer to treat HCC tumors.
Another embodiment provides a soluble T cell receptor. In one embodiment, the soluble TCR comprises an extracellular domain of a TCR polypeptide.
A. Genetically engineered T cell receptors
The disclosed TCR genes are from recombinant lv prime and peptide-enhanced AAD mice. Thus, technically, these TCRs are not "naturally occurring". In addition, after obtaining the V α and V β regions of the TCR, full length α and β chains were designed by using the identified V regions of α and β chains and the constant regions (C regions) of α and β chains of HLA-a2 mouse TCR specific for hgp 100. Thus, the entire TCR and its gene are not naturally occurring.
Protein sequences of mTCR-1, 2, 3,6, 8, 10, 11, 17 and 38 V.alpha.domains
One embodiment provides mTCR-1V α (TRAV7D-2 x 01/TRAJ30 x 01) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 2):
another embodiment provides mTCR-2V α (TRAV7D-2 x 01/TRAJ30 x 01) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 3):
comparing the mTCR-1. alpha. chain V region to the mTCR-2. alpha. chain, there are only 2 amino acid differences (bold and underlined).
Another embodiment provides mTCR-3V α (TRAV7D-2 x 01/TRAJ12 x 01) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 4):
there are a number of amino acid differences (bold and underlined) between the mTCR-1. alpha. chain and the mTCR-3. alpha. chain. In fact, the J segments are different (TRAJ 12 is used in mTCR-3V α instead of TRAJ 30).
One embodiment provides mTCR-6V α (TRAV7D-2 x 01/TRAJ30 x 01) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 5):
another embodiment provides mTCR-8V α (TRAV7D-2 x 01/TRAJ12 x 01) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 6):
there are a number of amino acid differences (bold and underlined) between the mTCR-6. alpha. chain and the mTCR-8. alpha. chain. In fact, the J segments are different (TRAJ 12 is used in mTCR-8V α instead of TRAJ 30).
Another embodiment provides mTCR-10V α (TRAV7D-2 x 01/TRAJ12 x 01) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 7):
comparing the mTCR-6. alpha. chain V region to the mTCR-8. alpha. chain, there is only a1 amino acid difference (bold and underlined).
Another embodiment provides mTCR-11V α (TRAV7D-2 x 01/TRAJ12 x 01) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 8):
comparing the mTCR-6. alpha. chain V region to the mTCR-8. alpha. chain, there are only 2 amino acid differences (bold and underlined).
One embodiment provides mTCR-17V α (TRAV7D-2 x 01/TRAJ12 x 01) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 9):
another embodiment provides mTCR-38V α (TRAV7D-2 x 01/TRAJ12 x 01) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 10):
in certain embodiments, the leader methionine and/or signal sequence is cleaved in the post-translationally modified protein.
Protein sequences of mTCR-1, 2, 3,6, 8, 10, 11, 17 and 38V beta domains
One embodiment provides mTCR-1, 2, 8, 11, 17 and 38V β domains (TRBV13-1 × 01/TRBJ2-4 × 01) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 11):
another embodiment provides a mTCR-3V β domain (TRBV13-1 x 01/TRBJ2-4 x 01) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 12):
there is only one amino acid difference (bold and underlined) between mTCR-1 and mTCR-2 with TCR-3V β.
Another embodiment provides a mTCR-6V β domain (TRBV13-1 x 01/TRBJ2-4 x 01) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 13):
there is only one amino acid difference from mTCR-1, 2, 8, and 11, only 2 amino acid differences from mTCR-3, and only 3 amino acid differences from mTCR-10.
Another embodiment provides a mTCR-10V β domain (TRBV13-1 x 01/TRBJ2-4 x 01) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 14):
there is a3 amino acid difference (bold and underlined) between mTCR-6V β and TCR-10V β.
In certain embodiments, the leader methionine and/or signal sequence is cleaved in the post-translationally modified protein.
3. Full-length protein sequences of mTCR-1, 2, 3,6, 8, 10, 11, 17, and 38 alpha chains
Another embodiment provides a mTCR-1. alpha. chain (TRAV 7D-2. multidot.01/TRAJ 30. multidot.01/TRAC) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 15):
the constant regions (C α) of the mTCR-1, 2, 3,6, 8, 10, and 11 α chains (bold) are identical, and they are identical to the hgp 100-specific TCR α chain C region from HLA-A2 Tg mice.
Another embodiment provides mTCR-2 α chain (TRAV7D-2 x 01/TRAJ30 x 01/TRAC) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 16):
another embodiment provides a mTCR-3. alpha. chain (TRAV7D-2 x 01/TRAJ12 x 01/TRAC) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 17):
another embodiment provides a mTCR-6. alpha. chain (TRAV 7D-2. multidot.01/TRAJ 30. multidot.01/TRAC) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 18):
another embodiment provides a mTCR-8. alpha. chain (TRAV 7D-2. multidot.01/TRAJ 12. multidot.01/TRAC) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 19):
another embodiment provides a mTCR-10. alpha. chain (TRAV 7D-2. multidot.01/TRAJ 12. multidot.01/TRAC) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 20):
another embodiment provides mTCR-11 α chain (TRAV7D-2 x 01/TRAJ12 x 01/TRAC) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 21):
one embodiment provides a mTCR-17 α chain (TRAV7D-2 x 01/TRAJ12 x 01/TRAC) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 22):
another embodiment provides a mTCR-38 a chain (TRAV7D-2 x 01/TRAJ12 x 01/TRAC) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO: 23):
in certain embodiments, the leader methionine and/or signal sequence is cleaved in the post-translationally modified protein.
In one embodiment, based on the nomenclature on IMGT and Uniprot websites, the mTCR α chain is considered to be characterized as follows:
1, M1-S20: leader sequence to be removed upon maturation of TCR alpha chain
2, Q21-S268: TCR-1, 2, 3 alpha chain
Q21-N132 (or D132): alpha chain V region
I133-S268: the same TCR α chain C region as HLA-A2 mouse TCR specific for hgp100 (DQ452619)
Q21-L242: extracellular domain of TCR alpha chain
S243-L263: transmembrane region of mature TCR alpha chain
R264-S268: intracellular region of mature TCR alpha chain
4. Full-length protein sequences of mTCR-1, 2, 3,6, 8, 10, 11, 17, and 38 beta chains
Another embodiment provides mTCR-1, 2, 8, 11, 17, and 38 beta strands (TRBV13-1 x 01/TRBJ2-4 x 01/TRBC1) having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 24):
the constant regions (C β) of the mTCR-1, 2, 3,6, 8, 10, 11, 17, and 38 β chains (bold) are identical, and they are identical to the hgp 100-specific TCR β chain C region from HLA-A2 Tg mice.
In certain embodiments, the leader methionine and/or signal sequence is cleaved in the post-translationally modified protein.
Another embodiment provides a mTCR-3. beta. chain (TRBV 13-1. multidot.01/TRBJ 2-4. multidot.01/TRBC 1) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:25)
Another embodiment provides a mTCR-6 beta strand (TRBV 13-1X 01/TRBJ 2-4X 01/TRBC1) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:26)
Another embodiment provides a mTCR-10. beta. chain (TRBV 13-1. multidot.01/TRBJ 2-4. multidot.01/TRBC 1) having 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:27)
In certain embodiments, the leader methionine and/or signal sequence is cleaved in the post-translationally modified protein.
In one embodiment, based on the nomenclature on IMGT and Uniprot websites, the mTCR α chain is characterized as follows:
1, M1-M17: leader sequence to be removed upon maturation of mTCR beta chain
E18-S304: mTCR beta chain
E18-L131: the V region of the mTCR beta chain (TRBV 13-1X 01/TRBJ 2-4X 01, wherein S113(TCR-1&2) or A113(TCR-3) -A116 is the D region)
E132-S304: mTCR beta chain C1 region of HLA-A mTCR specific for hgp100 (DQ452620)
E18-Y276: extracellular domain of mTCR beta chain
E277-M298: transmembrane region of mature mTCR beta chain
V299-S304: intracellular (topological) region of mature mTCR beta chain
Protein sequences of the CDR3 regions of the mTCR-1, 2, 3,6, 8, 10, 11, 17 and 38 alpha chains
One embodiment provides the mTCR-1. alpha. chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 28):
AASITNAYKVIFGKGTHLHVLPNIQNPE
another embodiment provides the mTCR-2. alpha. chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 29):
AASTVNAYKVIFGKGTHLHVLPNIQNPE
one embodiment provides the mTCR-3 a chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 30):
AASMAGGYKVVFGSGTRLLVSPDIQNPE
one embodiment provides the mTCR-6. alpha. chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 31):
AASMINAYKVIFGKGTHLHVLPNIQNPE
another embodiment provides the mTCR-8 a chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 32):
AASISGGYKVVFGSGTRLLVSPDIQNPE
one embodiment provides the mTCR-10. alpha. chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 33):
AASIVGGYKVVFGSGTRLLVSPDIQNPE
one embodiment provides the mTCR-11 a chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 34):
AASKTGGYKVVFGSGTRLLVSPDIQNPE
another embodiment provides the mTCR-17 a chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 35):
AASMTGGYKVVFGSGTRLLVSPDIQNPE
one embodiment provides the mTCR-38 a chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 36):
AATLTGGYKVVFGSGTRLLVSPDIQNPE
protein sequences of the CDR3 regions of the mTCR-1, 3,6 and 10 beta chains
One embodiment provides the mTCR-1 beta chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 37):
ASSDAGTSQNTLYFGAGTRLSVL
one embodiment provides the mTCR-3 beta chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 38):
ASSDAGTAQNTLYFGAGTRLSVL
another embodiment provides the mTCR-6 beta chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 39):
ASSDAGVSQNTLYFGAGTRLSVL
one embodiment provides the mTCR-10 beta chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 40):
ASSDHGTGQNTLYFGAGTRLSVL
extracellular domains of mTCR-1, 2, 3,6, 8, 10, 11, 17 and 38 alpha chains (for soluble TCR) (V alpha underlined)
The extracellular domains of the TCR α and β chains can form soluble TCRs. If they are labeled with a detectable marker (e.g., a fluorescent molecule), they can be used to detect HLA-A2/hAFP present on tumor cells158And (c) a complex. Labeled soluble TCRs have the potential to serve as diagnostic reagents to detect circulating tumor cells in blood and to determine cognate epitope presentation in tumor tissue, which is important for the context of successful use of such TCR-T cells for adoptive cell transfer therapy.
One embodiment provides a soluble mTCR-1. alpha. chain (Q21-L242) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 41):
another embodiment provides a soluble mTCR-2. alpha. chain (Q21-L242) having at least 90%, 95%, 99%, 100% sequence identity to (SEQ ID NO: 42):
another embodiment provides a soluble mTCR-3. alpha. chain (Q21-L242) having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 43):
another embodiment provides a soluble mTCR-6. alpha. chain (Q21-L242) having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 44):
another embodiment provides a soluble mTCR-8. alpha. chain (Q21-L242) having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 45):
another embodiment provides a soluble mTCR-10. alpha. chain (Q21-L242) having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 46):
another embodiment provides a soluble mTCR-11. alpha. chain (Q21-L242) having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 47):
one embodiment provides a soluble mTCR-17 α chain (Q21-L242) having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 48):
another embodiment provides a soluble mTCR-38 α chain (Q21-L242) having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 49):
the extracellular domains of TCR-1, 2, 3,6, 8, 10 and 11 beta chains (underlined is V beta)
One embodiment provides a soluble mTCR-1, 2, 8, 11 β chain (E18-Y276) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 50):
another embodiment provides a soluble mTCR-3. beta. chain (E18-Y276) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 51):
another embodiment provides a soluble mTCR-6 beta chain (E18-Y276) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 52):
another embodiment provides a soluble mTCR-10. beta. chain (E18-Y276) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO: 53):
9. designed mTCR fusion protein sequence
Fusion proteins
In another embodiment, a fusion protein comprising a first polypeptide domain and a second polypeptide is provided. The fusion protein can optionally comprise a targeting domain that targets a fusion protein-specific cell or tissue (e.g., a tumor cell or neovasculature associated with a tumor cell).
The fusion protein also optionally comprises a peptide or polypeptide linker domain that separates the first polypeptide domain from the antigen binding domain.
The fusion proteins disclosed herein have formula I:
N-R1-R2-R3-C
wherein "N" represents the N-terminus of the fusion protein, "C" represents the C-terminus of the fusion protein, "R1"is one of the disclosed α mTCR chains or fragments thereof. "R2"is a peptide/polypeptide linker Domain" R3"is a β mTCR chain or fragment thereof. In an alternative embodiment, R1Is a beta mTCR chain or a fragment thereof, R3Is an α mTCR chain.
Optionally, the fusion protein additionally contains a domain for dimerizing or multimerizing two or more fusion proteins. The domain used to dimerize or multimerize the fusion protein may be a separate domain or may be included in one of the other domains of the fusion protein.
The fusion protein may be dimerized or multimerized. Dimerization or multimerization may occur between or among two or more fusion proteins through a dimerization or multimerization domain. Alternatively, dimerization or multimerization of the fusion protein may occur by chemical crosslinking. The dimers or multimers formed may be homodimers/homomultimers or heterodimers/heteromultimers.
The modular nature of the fusion proteins and their ability to dimerize or multimerize in different combinations provides a rich choice for targeting molecules that function to enhance the immune response to the tumor cell microenvironment.
Another embodiment provides a fusion protein according to formula II,
N-R1-R2-R3-R2-R4-C
wherein "N" represents the N-terminus of the fusion protein, "C" represents the C-terminus of the fusion protein, "R1"is one of the disclosed α mTCR chains or fragments thereof. "R2"is a peptide/polypeptide linker Domain" R3"is a β mTCR chain or fragment thereof," R4 "is an anti-CD 3 single chain antibody. anti-CD 3 single chain antibodies are known in the art and are commercially available.
One embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:54)Designed byFusion protein of mTCR-1 (alpha chain underlined, P2A bold, beta chain double underlined):
the alpha chain is underlined, the beta chain is double underlined, and the P2A and furin cleavage sites (from US2016/0137715A1) are bolded.
Another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:55)Designed bymTCR-2 (alpha chain, P2A and beta chain)
Another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:56)Designed bymTCR-3(α chain, P2A, and β chain):
another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:57)Designed bymTCR-6 (alpha chain),P2A and β chain):
another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:58)Designed bymTCR-8(α chain, P2A, and β chain):
another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:59)Designed bymTCR-10(α chain, P2A, and β chain):
another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:60)Designed bymTCR-11(α chain, P2A, and β chain):
one embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:61)Designed bymTCR-17(α chain, P2A, and β chain):
one embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:62)Designed bymTCR-38(α chain, P2A, and β chain):
10. the designed nucleotide sequences of TCR-1, TCR-2, TCR-3, TCR-6, TCR-8, TCR-10, TCR-11, TCR-17 and TCR-38:
this sequence is based on the protein sequence described above and has codons optimized for expression in human cells.
One embodiment provides a nucleic acid sequence having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:63)Designed bymTCR-1(α underlined, P2A bold, β double underlined):
another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:64)Design ofmTCR-2(α underlined, P2A bold, β double underlined):
another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:65)Designed bymTCR-3(α underlined, P2A bold, β double underlined):
another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:66)Designed bymTCR-6 (alpha underlined, P2A plus)Coarse, β double underlined):
another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:67)Designed bymTCR-8(α underlined, P2A bold, β double underlined):
another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:68)Designed bymTCR-10(α underlined, P2A bold, β double underlined):
another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:69)Designed bymTCR-11(α underlined, P2A bold, β double underlined):
one embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:70)Designed bymTCR-17(α underlined, P2A bold, β double underlined):
another embodiment provides a polypeptide having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:71)Designed bymTCR-38(α underlined, P2A bold, β double underlined):
B. genetically engineered T cells
Another embodiment provides a genetically engineered CD8+ immune cell that expresses the disclosed mouse TCR genes to produce a TCR that specifically binds to hAFP or a fragment thereof expressed on the surface of a tumor cell. Preferably, the immune cell is a human T cell, such as a human cytotoxic T cell. The engineered human cytotoxic T cells may be autologous human cytotoxic T cells. The TCR gene can be codon optimized.
Another embodiment provides CD4+ immune cells, preferably human CD4+ immune cells, genetically engineered to express the disclosed mouse TCR genes. CD4+ cells include T helper cells, monocytes, macrophages and dendritic cells. The engineered human CD4+ immune cells may be autologous human T helper cells. The TCR gene can be codon optimized.
9 hAFPs identified in this study158Specific mouse TCRs capable of conferring CD4T cells with Tet binding158The ability of the tetramer suggests that they have high affinity and are not dependent on the help of CD 8. The role of CD4 in adoptive cell therapy is unclear. However, the production of IL-2 by CD4TCR-T cells in response to stimulation by HepG2 tumor cells may provide cytokines for maintaining T cell proliferation. Furthermore, CD4TCR-T cells showed low cytotoxicity against HepG2 tumor cells. Thus, although CD8 TCR-T may be a major player in killing hAFP + tumor cells, CD4TCR-T may provide help for T cell proliferation, which is useful for generating anti-tumor in vivoThe effect may be crucial.
It is believed that mouse TCRs can provide increased expression on the surface of human host cells compared to human TCRs. Mouse TCRs may also replace endogenous TCRs on the surface of human host cells more efficiently than exogenous human TCRs. However, to avoid repeated use of TCR-T cells to generate anti-TCR responses, humanization of mTCR may be required. Methods of humanizing mTCR are known in the art. See, for example, U.S. patents 5,861,155 and 5,859,205, WO2007/131092, EP0460167, and Davis et al, ClinCancer res, vol 16, page 5852 and 5861, 2010, which are incorporated herein by reference in their entirety. In one embodiment, the disclosed mTCR genes are humanized prior to introduction into T cells or administration to a human subject.
C.T cell hybridoma
Another embodiment provides a T cell hybridoma expressing the disclosed mTCR polypeptide. For example, the hybridoma may be a mouse CD8+ Tet to be sorted158+ cells with BW-Lyt2.4 cells lacking TCR. alpha. and. beta. chains and were selected as described (He., Y et al, J Immunol, Vol.174, p.3808-3817, 2005). Methods of making hybridomas are provided in the examples.
D. Antibodies
One embodiment provides antibodies that specifically bind to the disclosed mTCR proteins (e.g., SEQ ID NOS: 2-62). Suitable antibodies can be prepared by one skilled in the art. Thus, an antibody or antigen-binding fragment may be an agonist or antagonist of mTCR, or simply specifically bind to mTCR or a polypeptide thereof.
In some embodiments, the disclosed antibodies and antigen-binding fragments thereof immunospecifically bind to mTCR or a polypeptide thereof (e.g., any of SEQ ID NOS: 2-62). In some embodiments, the antibody binds to the extracellular domain of mTCR (SEQ ID NOS: 41-53).
For example, molecules that immunospecifically bind to the disclosed mTCR polypeptides are provided:
(I) arranged on the surface of a cell (particularly a living cell); or
(II) is arranged on the surface of a cell (in particular a living cell) in an endogenous concentration;
also provided are compositions that immunospecifically bind to a soluble endogenous mTCR polypeptide. In some embodiments, the molecule reduces or prevents the soluble mTCR polypeptide from binding or otherwise interacting with its ligand.
The antibody or antigen-binding fragment thereof can be prepared using any suitable method known in the art, such as those discussed in more detail below.
1. Human and humanized antibodies
Antibodies that specifically bind to the disclosed mTCR polypeptides can be human or humanized. Many non-human antibodies (e.g., those derived from mouse, rat, or rabbit) are naturally antigenic in humans and, therefore, cause an adverse immune response when administered to humans. Thus, use of a human or humanized antibody in these methods helps to reduce the chance that administration of the antibody to a human will cause an adverse immune response.
Transgenic animals (e.g., mice) that are capable of producing a complete human antibody repertoire upon immunization without the production of endogenous immunoglobulins can be used. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (j (h)) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays in such germline mutant mice will result in the production of human antibodies upon antigen challenge.
Optionally, the antibody is produced in other species and "humanized" for administration in humans. Humanized forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab ', F (ab')2Or other antigen binding subsequences of antibodies). Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient antibody are substituted by residues from a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some cases, Fv framework residues of the human immunoglobulin are substituted with corresponding non-human residues. HumanizationThe antibody may also comprise residues that are present in neither the recipient antibody nor the imported CDR or framework sequences. Typically, a humanized antibody will comprise substantially all of at least one variable domain, and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise an immunoglobulin constant region (Fc), typically at least a portion of a constant region of a human immunoglobulin.
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Antibody humanization techniques typically involve the use of recombinant DNA techniques to manipulate DNA sequences encoding one or more polypeptide chains of an antibody molecule. Humanization can be essentially performed by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Thus, a humanized form of a non-human antibody (or fragment thereof) is a chimeric antibody or fragment in which substantially less than the entire human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
To reduce antigenicity, it is important to select the human variable domains (both light and heavy) used to make the humanized antibody. According to the "best fit" method, sequences of rodent antibody variable domains are screened against an entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then accepted as the human Framework (FR) of the humanized antibody. Another approach uses specific frameworks derived from the consensus sequence of all human antibodies of a specific subset of light or heavy chains. The same framework can be used for several different humanized antibodies.
It is further important to humanize antibodies while retaining high affinity for antigens and other advantageous biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs can be used to illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected and combined from the consensus and introduced sequences to achieve desired antibody characteristics, such as increased affinity for one or more target antigens. Generally, CDR residues are directly and primarily involved in substantially affecting antigen binding.
The antibody may be bound to a substrate or labeled with a detectable moiety, or both. Detectable moieties contemplated by the compositions of the present invention include fluorescent, enzymatic and radioactive labels.
2. Single chain antibody
The antibody that specifically binds to the disclosed mTCR polypeptide can be a single chain antibody. Methods for producing single chain antibodies are well known to those skilled in the art. Single chain antibodies are formed by fusing the variable domains of the heavy and light chains together using a short peptide linker to reconstitute the antigen binding site on a single molecule. Without significantly disrupting antigen binding or binding specificity, single chain antibody variable fragments (scfvs) have been developed in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain via a 15 to 25 amino acid peptide or linker. The linker is selected to allow the heavy and light chains to bind together in their proper conformational orientation. These fvs lack the constant region (Fc) found in the heavy and light chains of natural antibodies.
3. Monovalent antibodies
The antibody that specifically binds to the disclosed mTCR polypeptide can be a monovalent antibody. Monovalent antibodies can be prepared using in vitro methods. The antibodies can be digested to produce fragments thereof using conventional techniques known in the artIn particular Fab fragments. For example, digestion can be performed using papain. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each having a single antigen binding site and a residual Fc fragment. Pepsin treatment produced a protein called F (ab')2A fragment of a fragment that has two antigen binding sites and is still capable of cross-linking antigens.
The Fab fragment produced by antibody digestion also contains the constant domain of the light chain and the first constant domain of the heavy chain. Fab' fragments differ from Fab fragments by the addition of residues at the carboxy terminus of the heavy chain domain, including one or more cysteines from the antibody hinge region. F (ab')2A fragment is a bivalent fragment comprising two Fab' fragments linked by disulfide bonds of the hinge region. Fab '-SH is the designation herein for Fab', where the cysteine residues of the constant domains carry free thiol groups. Antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
4. Hybrid antibodies
The antibody that specifically binds to the disclosed mTCR polypeptide can be a hybrid antibody. In hybrid antibodies, one heavy and light chain pair is homologous to the heavy and light chain pair found in an antibody directed to one epitope, while the other heavy and light chain pair is homologous to the heavy and light chain pair found in an antibody directed to another epitope. This results in the property of having a multifunctional valency, i.e. the ability to bind at least two different epitopes simultaneously. Such hybrids can be formed by fusion of hybridomas producing antibodies of the respective components or by recombinant techniques. Such hybrids can of course also be formed using chimeric chains.
5. Conjugates or fusions of antibody fragments
The targeting function of the antibody can be used therapeutically by coupling the antibody or fragment thereof to a therapeutic agent. Such coupling of an antibody or fragment (e.g., at least a portion of an immunoglobulin constant region (Fc)) to a therapeutic agent can be achieved by preparing an immunoconjugate or by preparing a fusion protein comprising the antibody or antibody fragment and the therapeutic agent.
Such coupling of the antibody or fragment to the therapeutic agent can be achieved by preparing an immunoconjugate, or by preparing a fusion protein, or by linking the antibody or fragment to a nucleic acid (such as an siRNA), all of which include the antibody or antibody fragment and the therapeutic agent.
In some embodiments, the antibody is modified to alter its half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that it is present in the circulation or at the treatment site for a longer period of time. For example, it may be desirable to maintain the titer of the antibody in the circulation or at the site to be treated for an extended period of time. Antibodies can be engineered with Fc variants that extend half-life, e.g., using XtendTMAntibody half-life extension techniques (xenocor, monprovia, CA). In other embodiments, the half-life of the anti-DNA antibody is reduced to reduce potential side effects. The disclosed conjugates can be used to modify a given biological response. The drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin.
The disclosed antibodies and mTCR polypeptides may be conjugated or linked to one or more detectable labels. The disclosed antibodies and mTCR polypeptides can be linked to at least one reagent to form a detection conjugate. To enhance the efficacy of a molecule as a diagnostic agent, it is typically linked or covalently bound or complexed with at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one reporter molecule. A reporter is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules that have been conjugated to an antibody or polypeptide include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, and/or ligands, such as biotin.
E. Preparation
The disclosed antibodies, fusion proteins, and mTCR polypeptides may be formulated into pharmaceutical compositions. Pharmaceutical compositions comprising the antibody, fusion protein or mTCR polypeptide may be administered by parenteral (intramuscular, intraperitoneal, Intravenous (IV) or subcutaneous injection), transdermal (passive or using iontophoresis or electroporation) or transmucosal (nasal, vaginal, rectal or sublingual) routes of administration or using a biodigestible insert, and may be formulated into dosage forms suitable for each route of administration.
In some in vivo methods, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dose sufficient to treat, inhibit or alleviate one or more symptoms of the disease being treated or to otherwise provide a desired pharmacological and/or physiological effect. The precise dosage will vary depending on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being performed.
As further studies are conducted for the disclosed compositions, information will emerge regarding appropriate dosage levels for treating various conditions in various patients, and the skilled artisan will be able to determine the correct dosage in view of the treatment context, age and general health of the recipient. The selected dosage depends on the desired therapeutic effect, the route of administration and the desired treatment time. For the disclosed immunomodulators, administration to a mammal is generally at a dosage level of 0.001 to 20mg/kg body weight per day. Generally, for intravenous injection or infusion, the dosage may be lower.
In certain embodiments, the disclosed compositions are administered topically, e.g., by direct injection to the site to be treated. Typically, injection causes an increase in the local concentration of the composition, which is higher than that achievable by systemic administration.
The composition may be combined with a matrix as described above to help increase the local concentration of the polypeptide composition by reducing passive diffusion of the polypeptide from the site to be treated.
1. Formulations for parenteral administration
In some embodiments, the compositions disclosed herein, including those comprising peptides and polypeptides, are administered as an aqueous solution by parenteral injection. The formulation may also be in the form of a suspension or emulsion. Generally, a pharmaceutical composition is provided comprising an effective amount of a peptide or polypeptide and optionally a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and/or carrier. Such compositions optionally include one or more of the following: diluent, sterile water, various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength buffered saline; and additives such as detergents and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite) and preservatives (e.g., thimerosal, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of nonaqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil and corn oil), gelatin, and injectable organic esters (such as ethyl oleate). The formulation may be lyophilized and re-solubilized/resuspended prior to use. The formulation may be sterilized, for example, by filtration through a bacterial-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.
2. Oral administration preparation
In embodiments, the composition is formulated for oral delivery. Oral solid dosage forms are generally described in Remington's Pharmaceutical Sciences, 18 th edition, 1990(Mack Publishing co. easton pa.18042) chapter 89. Solid dosage forms include tablets, capsules, pills, lozenges or troches, cachets, pills, powders or granules, or incorporating the material into a granular formulation or liposomes of polymeric compounds such as polylactic acid, polyglycolic acid, and the like. Such compositions can affect the disclosed physical state, stability, rate of in vivo release, and rate of in vivo clearance. See, e.g., Remington's Pharmaceutical Sciences, 18 th edition, (1990, Mack Publishing Co., Easton, Pa.18042) page 1435-1712, which is incorporated herein by reference. The compositions may be prepared in liquid form, or may be in the form of a dry powder (e.g., lyophilized). The compositions may be formulated using liposome or proteoid encapsulation. Encapsulation with liposomes can be used, and liposomes can be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). See also Marshall, k.in: modern pharmaceuticals, edited by g.s.banker and c.t.rhodes, chapter 10, 1979. Typically, the formulation will include the peptide (or chemically modified form thereof) and inert ingredients that protect the peptide in the gastric environment and release the biologically active substance in the intestine.
The agents may be chemically modified to render the oral delivery of the derivatives effective. Generally, the chemical modification envisaged is the attachment of at least one moiety to the component molecule itself, wherein the moiety allows absorption from the stomach or intestine into the blood stream or directly into the intestinal mucosa. It is also desirable to increase the overall stability of one or more components and increase circulation time in vivo. Pegylation is an exemplary chemical modification for pharmaceutical use. Other moieties that may be used include: propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1, 3-dioxolane and poly-1, 3, 6-trioxahicyclo [ see, e.g., Abuchowski and Davis (1981), "Soluble Polymer-enzyme Adducts", published in Enzymes as Drugs, Hounberg and Roberts, ed. (Wiley-Interscience: New York, N.Y.) p.367-383; and Newmark et al, (1982) J.appl.biochem. Vol.4, pp.185-189 ].
Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other components, including inert diluents, adjuvants (such as wetting, emulsifying, and suspending agents), and sweetening, flavoring, and perfuming agents.
Controlled release oral formulations may be desirable. The agent may be incorporated into an inert matrix, such as a glue, which allows release by diffusion or leaching mechanisms. Slowly degrading substrates may also be incorporated into the formulation. Another form of controlled release is based on the Oros therapeutic system (Alza Corp.), i.e. the drug is encapsulated in a semi-permeable membrane which, due to osmotic action, allows water to enter and push the drug out through a single small opening.
For oral formulations, the site of release may be the stomach, small intestine (duodenum, jejunum or ileum) or large intestine. In some embodiments, by a protective agent (or derivative) or byReleasing the agent (or derivative) outside the gastric environment, such as in the intestine, for release would avoid the deleterious effects of the gastric environment. To ensure complete gastric resistance, a coating that is impermeable at least at pH 5.0 is essential. Examples of more common inert ingredients used as enteric coatings are: cellulose Acetate Trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, vinyl acetate phthalate (PVAP), Eudragit L30DTM、AquatericTMCellulose Acetate Phthalate (CAP) and Eudragit LTM、Eudragit STMAnd ShellacTM. These coatings can be used as mixed films.
3. Formulations for topical application
The disclosed immunomodulators can be administered topically. Although topical administration is particularly effective when applied to the pulmonary, nasal, buccal (sublingual, buccal), vaginal or rectal mucosa, it is not ideal for most peptide formulations.
When the composition is delivered as an aerosol or spray dried particles (aerodynamic diameter less than about 5 microns), the composition may be delivered to the lungs and cross the lung intraepithelial layer into the blood stream simultaneously with inhalation.
A variety of mechanical devices designed for pulmonary delivery of therapeutic products may be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are Ultravent atomizers (Mallinckrodt inc., st. louis, Mo.), Acorn II atomizers (Marquest Medical Products, Englewood, Colo.), Ventolin metered inhalers (Glaxo inc., Research Triangle Park, n.c.), and spinoler powder inhalers (fish corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin powder formulations that have been approved or that the technology can be applied to the formulations described herein in clinical trials.
Formulations for mucosal administration are typically spray-dried drug particles, which may be incorporated into tablets, gels, capsules, suspensions or emulsions. Standard pharmaceutical excipients are available from any formulator.
Transdermal preparations can also be prepared. These are typically ointments, lotions, sprays or patches, all of which can be prepared using standard techniques. Transdermal formulations may need to contain a penetration enhancer.
4. Controlled delivery polymer matrix
The fusion proteins, mTCR polypeptides, and antibodies disclosed herein can also be administered in a controlled release formulation. Controlled release polymer devices can be made for systemic long term release after implantation of the polymer device (rod, column, membrane, disc) or injection (microparticles). The matrix may be in the form of microparticles such as microspheres, in which the agent is dispersed in a solid polymer matrix or microcapsules, in which the material of the core is different from the material of the polymeric shell, and the peptide is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules may be used interchangeably. Alternatively, the polymer may be cast as a sheet or film (ranging from nanometers to four centimeters), may be a powder produced by grinding or other standard techniques, and may even be a gel, such as a hydrogel.
Non-biodegradable or biodegradable matrices can be used to deliver the fusion polypeptide, antibody or mTCR polypeptide or nucleic acid encoding them, although biodegradable matrices are preferred in some embodiments. These may be natural or synthetic polymers, but in some embodiments synthetic polymers are preferred due to better characterization of degradation and release characteristics. The polymer is selected based on the time period that release is desired. In some cases, linear release may be most useful, but in other cases, pulsed release or "batch release" may provide more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% by weight water), and may optionally be crosslinked with a multivalent ion or polymer.
These matrices may be formed by solvent evaporation, spray drying, solvent extraction and other methods well known to the skilled artisan. The bio-digestible microspheres may be prepared using any method developed for the preparation of drug delivery microspheres, such as, for example, Mathiowitz and Langer, J., Controlled Release, Vol.5, pp.13-22, 1987; mathiowitz et al, Reactive Polymers, Vol.6, p.275-; and Mathiowitz et al, J.appl.Polymer Sci, Vol.35, p.755-774, 1988.
These devices can be formulated for local delivery to treat the area of implantation or injection (typically providing a much smaller dose than required for systemic treatment) or for systemic delivery. These may be implanted or injected subcutaneously into muscle, fat, or swallowed.
Method of use
In certain embodiments, the disclosed antibodies, mTCR polypeptides, and engineered immune cells can be used to detect or treat cancer. Preferred cancers to be detected, diagnosed or treated include hepatocellular carcinoma or any other cancer expressing hAFP.
A. Adoptive transfer
In one embodiment, immune cells are engineered to express the disclosed mtcrs. The immune cell is a T cell, preferably a human T cell. The cells may be autologous or heterologous. Cells are typically transduced in vitro. The transduced cells can optionally be expanded in vitro to obtain a large population of transduced cells that can be administered to a subject in need thereof. Such subjects typically have or are believed to have a cancer or tumor that expresses hAFP. The T cells may be CD8+ or CD4+ cells. The transduced cells can be administered to a subject in one or more doses.
Adoptive transfer can be used in conjunction with other cancer treatment therapies. Such additional therapies include chemotherapy, radiation therapy, and surgery or combinations thereof. Adoptive transfer of TCR-T can be used in conjunction with cancer vaccines, thereby allowing further expansion of TCR-T in vivo to reduce the number of TCR-T cells transferred and associated toxicity.
One embodiment provides a method of reducing tumor burden in a subject in need thereof by administering an engineered T cell expressing one of the disclosed mtcrs, wherein the engineered mTCR cell inhibits or reduces a tumor expressing hAFP. The subject may optionally be treated with a combination therapy for cancer. The preferred cancer to be treated is hepatocellular carcinoma.
B. Detection or diagnosis
The disclosed antibodies or mTCR polypeptides can be labeled as described above. The labeled polypeptide can be applied to a cancer cell sample, wherein specific binding of the labeled polypeptide to the cancer cell sample is indicative of an hAFP-specific cancer, including hepatocellular carcinoma.
Detection of the marker can optionally be quantified to determine the number of cancer cells in the sample. The sample is typically a liver tissue sample, but may include a blood or serum sample.
Compositions and kits
Compositions and kits are also provided. Such kits may include containers, each container having one or more of the various reagents (typically in concentrated form) utilized in the method, including, for example, one or more binding agents (antibodies) already attached to the label or optionally having reagents for coupling the binding agent to the antibody (as well as the label itself), buffers, and/or reagents and instruments for isolation (optionally by microdissection) to support the practice of the invention. Typically, a label or instructions describing the components of the kit or a set of instructions for use in the ligand detection methods of the invention will also be included, wherein the instructions may be associated with the package insert and/or package of the kit or components thereof.
Additional embodiments provide immunoassay kits for use with the immunoassay methods described herein.
Since antibodies or mTCR polypeptides are commonly used to detect hAFPs, antibodies and mTCR polypeptides will typically be included in the kit. Thus, the immunoassay kit will include in a suitable container means a first antibody that binds to the mTCR polypeptide and/or optionally an immunoassay reagent.
The immunodetection reagents of the kit can take any of a variety of forms, including those detectable labels associated with and/or linked to a given antibody. Detectable labels associated and/or attached to secondary binding ligands are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.
Other suitable immunodetection reagents for use in the present kit include two-component reagents comprising a second antibody having binding affinity for the first antibody and a third antibody having binding affinity for the second antibody, the third antibody being linked to a detectable label. As described herein, a number of exemplary labels are known in the art, and/or all such labels can be suitably used in conjunction with the present invention.
The kit can also include a therapeutic agent for treating cancer, such as an engineered immune cell that expresses the disclosed mTCR polypeptide.
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