Multispecific 2a3 antibody, nucleic acid molecule encoding the antibody, vector comprising the nucleic acid molecule, construct comprising a car with the antibody, and their use in car-t cell therapy for treating solid tumors

Abstract

The invention relates to the 2A3 antibody characterized in that it exhibits multispecificity toward CEACAM family proteins selected from the group comprising CEACAM1, CEACAM3, CEACAM5, and CEACAM6. Furthermore, the invention relates to a nucleic acid molecule encoding the antibody according to the invention, a vector comprising the said nucleic acid molecule, and a CAR construct with the said antibody. A further subject of the invention is the 2A3 antibody for use in CAR-T therapy for treating tumors exhibiting overexpression of a protein selected from CEACAM1, CEACAM3, CEACAM5, CEACAM6, or all of the foregoing simultaneously.

Description

DescriptionMULTISPECIFIC 2A3 ANTIBODY, NUCLEIC ACID MOLECULE ENCODING THE ANTIBODY, VECTOR COMPRISING THE NUCLEIC ACID MOLECULE, CONSTRUCT COMPRISING A CAR WITH THE ANTIBODY, AND THEIR USE IN CAR-T CELL THERAPY FOR TREATING SOLID TUMORS

[0001] The subject of the invention is a 2A3 antibody that is multispecific toward particular proteins from the CEACAM family. A further subject of the invention is a nucleic acid molecule encoding said antibody, a vector comprising said nucleic acid molecule, and a construct encoding a CAR that comprises said antibody. Furthermore, the invention relates to the use of the said antibody, including its CAR-encoding construct, in therapy of solid tumors.Technical Field

[0002] Human carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family (CEA-related cell adhesion molecules) comprises adhesion molecules belonging to the immunoglobulin superfamily (IgSF). CEACAM antigens (CD66) have been included in the IgSF superfamily due to the presence in their structure of variable and constant immunoglobulin-like domains. The CEACAM family consists of 12 members, including CEACAM1, CEACAM3-CEACAM8, CEACAM16, and CEACAM18-21. Members of the CEACAM family differ in the number of Ig-like domains, degree of glycosylation, presence of isoforms, and tissue distribution.Background Art

[0003] W02012040824A1 discloses anti-CEACAM6 antibodies and their use for treating cancers, as well as methods of blocking CEACAM6 and reducing its invasiveness, decreasing proliferation, cell invasion, and MMP-9 activity; and reducing the ability of cancer cells to promote angiogenesis. That document discloses that an antibody containing CDR1, CDR2, and CDR3 regions of specific amino acid sequences is exclusively specific for CEACAM6.

[0004] Jancewicz et al., in “New CEACAM-targeting 2A3 single-domain antibodybased chimeric antigen receptor T-cells produce anticancer effects in vitro and in vivo. Cancer Immunol Immunother. 2024 Jan 27;73(2):30. doi: 10.1007 / s00262-023-03602-4,” reported that the 2A3 antibody is bispecific, i.e., it binds exclusively to CEACAM6 as well as CEACAM5. This article indicates that although CEACAM6 and CEACAM5 are members of the same family, cytotoxic effects occur exclusively for these two family members.

[0005] Unexpectedly, it turned out that the 2A3 antibody exhibits multispecificity toward other proteins of the CEACAM family, i.e., toward proteins selected from the group comprising CEACAM 1, CEACAM3, CEACAM5, and CEACAM6. Thus, the present invention provides the 2A3 antibody which, by recognizing various proteins from the CEACAM family, finds application in CAR-T therapy for the treatment of cancer, inter alia by blocking tumor escape.Summary of Invention

[0006] The subject of the invention is a 2A3 antibody characterized in that it exhibits multispecificity toward CEACAM family proteins selected from the group comprising CEACAM 1, CEACAM3, CEACAM5, and CEACAM6.

[0007] Preferably, the CEACAM family proteins comprise an epitope containing the amino acid sequence LFGYSWYKG.

[0008] Preferably, the CEACAM family proteins comprise an epitope containing the amino acid sequence GYSWYKG.

[0009] Preferably, the antibody is fused to an Fc fragment.

[0010] A further subject of the invention is a nucleic acid molecule encoding the antibody according to the invention.

[0011] Another subject of the invention is a vector comprising the nucleic acid molecule according to the invention.

[0012] Another subject of the invention is a CAR-T construct comprising the antibody according to the invention.

[0013] Another subject of the invention is the 2A3 antibody, according to the invention, for use in therapy.

[0014] Preferably the therapy is selected from the group comprising cellular immunotherapy, treatment of autoimmune diseases, a therapy in which the 2A3 antibody is conjugated to a cytotoxic drug or a PET tracer, radioimmunotherapy, photoimmunotherapy, a therapy employing a bispecific or trispecific activator of T lymphocytes or natural killer (NK) cells, an immunomodulatory therapy using 2A3 as a blocking antibody or as an antibody with a silenced Fc, a targeted drug-delivery and diagnostic therapy in which 2A3 is linked to liposomes, gold nanoparticles, or polymeric micelles, or is labelled with a dye for intraoperative tumor visualization, or is fused to a viral protein for infection of CEACAM-positive cells.

[0015] Another subject of the invention is the 2A3 antibody, according to the invention, for use in CAR-T therapy for treating tumors exhibiting overexpression of a protein selected from CEACAM1, CEACAM3, CEACAM5, CEACAM6, or all of the foregoing simultaneously.

[0016] Another subject of the invention is the 2A3 antibody, according to the invention, for use in CAR-T therapy for treating tumors exhibiting overexpression of a protein selected from CEACAM1, CEACAM3, CEACAM5, CEACAM6, or all of the foregoing simultaneously.

[0017] Preferably, the tumor does not exhibit overexpression of CEACAM6.

[0018] Preferably, the tumor is a solid tumor selected from the group comprising cancer of the pancreas, breast, gallbladder and biliary tract, colon, lung, urinary bladder, uterus, prostate, gastrointestinal tract, thyroid, testis, head and neck, and melanoma.

[0019] Preferably, proteins selected from CEACAM1, CEACAM3, and CEACAM5 comprise an epitope containing the amino acid sequence LFGYSWYKG.

[0020] Preferably, the 2A3 antibody according to the invention is for use in CAR-T therapy for treating tumors, wherein patient selection includes confirmation of the absence of CEACAM6 overexpression in tumor cells and the overexpression in tumor cells of CEACAM1, CEACAM3, and CEACAM5 proteins comprising an epitope containing the amino acid sequence LFGYSWYKG.

[0021] Preferably, the 2A3 antibody for use according to the invention is characterized in that the tumor is a solid tumor selected from the group comprising cancer of the pancreas, breast, gallbladder and biliary tract, colon, lung, urinary bladder, uterus, prostate, gastrointestinal tract, thyroid, testis, head and neck, and melanoma. Advantageous Effects of Invention

[0022] An advantage of the antibody according to the invention is its multispecificity toward CEACAM family proteins, which enables expansion of previously known therapeutic targets, while the said antibody that binds an epitope with the amino acid sequence GYSWYKG should not lead to a cytotoxic effect against healthy cells, such as cells containing the protein ZCCHC3 in which the said amino acid sequence occurs, since that protein is intracellular.Brief Description of Drawings

[0023] The subject of the invention is explained in greater detail in the embodiments and illustrated with reference to the drawings, wherein:

[0024] [Fig.l] shows the results of flow cytometry (FC) analysis of staining of MCF7 cells with knockout of CEACAM6, stained with the 2A3-Fc antibody,

[0025] [Fig.2a]-b show Western blot (WB) and qPCR (bar plot) results confirming gene expression and the presence of CEACAM proteins in respective clonal lines obtained from HEK293T cells subjected to viral transduction; the 2A3 antibody recognizes CEACAM 1 and CEACAM3 by WB, whereas it does not recognize CEACAM7 (boxed),

[0026] [Fig-3] shows the results of flow cytometry analysis of staining with 2A3-Fc of cells with overexpression of individual CEACAM proteins; the curve with the maximum peak shifted toward higher values on the x-axis represents the overexpression line; the second curve corresponds to the control cell line (HEK293T Ctrl or MDA-MB-231 Ctrl, respectively),

[0027] [Fig.4] shows the results of sequence alignment for CEACAM family proteins (-1,-3, -5, -6, -7); the darker the shade, the greater the sequence similarity.Description of Embodiments

[0028] The antibody according to the invention, i.e., the 2A3 antibody, is characterized in that it comprises: CDR1 with the sequence GRTNSVYTMG (SEQ ID NO: 1), CDR2 with the sequence IMWGAGTNTHYADSVKG (SEQ ID NO: 2), and CDR3 with the sequence AANRGIPIAGRQYDY (SEQ ID NO: 3). The antibody according to the invention is a single-domain antibody.

[0029] The subject of the invention is useful in the therapy of cancers in which the tumor is a solid tumor selected from the group comprising cancer of the pancreas, breast, gallbladder and biliary tract, colon, lung, urinary bladder, uterus, prostate, gastrointestinal tract, thyroid, testis, head and neck, and melanoma.

[0030] The invention finds use in treating cancers in which overexpression of the proteins CEACAM 1, CEACAM3, CEACAM5, and CEACAM6 is observed.

[0031] Preferably, the invention also finds use in treating cancers that do not exhibit overexpression of CEACAM6, i.e., that exhibit overexpression of one, two, or all of the proteins selected from CEACAM1, CEACAM3, and CEACAM5. This includes, for example, melanoma, where elevated levels of CEACAM 1 are observed, but not CEACAM6, -5, or -3; or, for example, gallbladder cancers, where increased level of CEACAM5 is observed.

[0032] In one aspect of the invention, the vector comprising the nucleic acid molecule encoding the antibody according to the invention is a vector derived from retroviruses, such as lentiviruses. Lentiviral vectors have an added advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. Expression of natural or synthetic nucleic acids encoding a CAR is typically achieved by operably linking the nucleic acid encoding the CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. Vectors may be suitable for replication and integration in eukaryotes. Typical cloning vectors comprise transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

[0033] In one aspect of the invention, the nucleic acid molecule can be cloned into many types of vectors. For example, the nucleic acid molecule can be cloned into a vector including, without limitation, a plasmid, phagemid, phage derivative, animal virus, and cosmid. Vectors include expression vectors, replication vectors, vectors for generating probes, and sequencing vectors.

[0034] An isolated or purified 2A3 antibody can be expressed fused to an Fc fragment; in particular, the Fc fragment can be mouse Fc2b or human Fcl.

[0035] In one aspect of the invention, the antibody according to the invention forms a protein construct with a CAR - chimeric antigen receptor. CARs are introduced into modified T lymphocytes (CAR-T). The CAR-T therapy according to the present invention is used for treating solid tumors.

[0036] Examples illustrating, but not limiting, the invention are presented below.Examples

[0037] Example 1

[0038] Assessment of 2A3 binding to a cell line lacking CEACAM6.

[0039] In an MCF7 cell line with knockout of CEACAM6, staining was performed with the 2A3 protein fused to a human Fc fragment (2A3-Fc) and a secondary anti-human antibody labeled with DyLight488. The percentage of stained cells was assessed by FC. In two replicates, a population of MCF7 cells was obtained to which the 2A3 antibody bound (-25%) despite the absence of CEACAM6 expression.

[0040] Example 2

[0041] Binding of the 2A3 antibody to cell lines with overexpression of individual CEACAM family proteins.

[0042] Cell lines were generated: MDA-MB-231 (negative control) with overexpression of CEACAM6 and CEACAM5, and HEK293T (also a negative control) with overexpression of CEACAM1, CEACAM3, and CEACAM7 (each separately). Expression of these proteins was confirmed by three methods - qPCR, Western blot, and FC.

[0043] Binding of the 2A3-Fc protein to the above cell lines was examined. All cell lines except the HEK293T line with overexpression of CEACAM7 (CEACAM7ox) were stained by 2A3-Fc, indicating recognition of these proteins by the 2A3 antibody.

[0044] Example 3

[0045] Cytotoxicity of CAR-T cells with a CAR based on 2A3.

[0046] A cytotoxicity analysis was performed on the above cell lines using CAR-T cells with a CAR based on the 2A3 antibody. For all lines except the HEK293T line overexpressing CEACAM7, a cytotoxic effect of 2A3-CAR-T cells was observed.

[0047] The studies performed confirm ([Fig.4]) that the 2A3 antibody binds to CEACAM1, CEACAM3, and CEACAM5 proteins because it binds an epitope common to these proteins and having the amino acid sequence LFGYSWYKG at positions 62-70 (SEQ ID NO: 4). Moreover, the antibody binds to CEACAM1, CEACAM3, CEACAM5, and CEACAM6 because it binds an epitope common to these proteins and having the amino acid sequence GYSWYKG at positions 64-70 (SEQ ID NO: 5).

[0048] The 2A3 antibody, including a CAR-encoding construct with the 2A3 antibody, is thus useful in reducing tumors, and consequently in therapy of solid tumors. In particular, usefulness is observed for cancers of the pancreas, breast, gallbladder and biliary tract, colon, lung, urinary bladder, uterus, prostate, gastrointestinal tract, thyroid, testis, head and neck, and melanoma.

[0049] Example 4

[0050] Analysis of expression of proteins from the group of CEACAM1, CEACAM3, CEACAM5, and CEACAM6 in lung cancer lines (NCI-H23, NCI-H460, NCI-H647), an ovarian cancer line (SKOV-3), and a CEACAM-positive control line (BxPC-3), and a cytotoxicity test of 2A3-CAR-T cells against cells of these lines.

[0051] A cytotoxicity test of 2A3-CAR-T cells and control cells was performed against the cancer cell lines BxPC-3 (positive control), NCI-H23, NCI-H460, NCI-H647, and SKOV-3. The study was conducted by impedance measurement using an RTCA xCELLanalyser instrument (Agilent). A cytotoxic effect was found against the BxPC-3 line and the NCI-H647 line, and a minor effect in the case of the NCI-H23 line. The result is consistent with the results of transcript level analysis for proteins in these lines, based on data available in The Human Genome Atlas. Elevated expression of proteins from the group CEACAM1, CEACAM3, CEACAM5, and CEACAM6 in the cell lines can also be confirmed by qPCR using the AACt method and obtaining a result >1 compared with a low-expression line (result <1), or by Western blot and flow cytometry by obtaining positive staining with antibodies specific to the said proteins (no staining in lines without expression).

[0052] In the BxPC-3 line, which is treated as a positive control, very high expression of CEACAM1, CEACAM5, and CEACAM6 (in total >2000) was found, as well as very rapid cell death in co-culture with 2A3-CAR-T lymphocytes. In the NCI-H647 line, the total expression of the said proteins was lower and cell death occurred more slowly. In NCI-H23 cells, the number of transcripts of the said proteins is the lowest, and the cytotoxic effect is visible, though minor.

[0053] In lines in which no expression of proteins from the group CEACAM1, CEACAM3, CEACAM5, and CEACAM6 was found (total pTPM < 0.5), i.e., MDA-MB-231, MIA PaCa-2, and SK-OV-3, no cytotoxic effect was observed.

[0054] The results of the experiment and data analysis clearly indicate that 2A3-CAR-T recognizes only cells in which expression of proteins from the group CEACAM1, CEACAM3, CEACAM5, and CEACAM6 has been confirmed. In cells in which there is no expression of proteins from this group, no cytotoxic effect was found. This confirms the specificity of the 2A3 antibody and 2A3-CAR-T cells toward the proposed group of antigens. In addition, the results suggest that the cytotoxic response of 2A3-CAR-T cells may depend on the level of these proteins on the surface of cancer cells.

[0055] At the same time, the data presented indicate that the 2A3 antibody can be used in various cancer types, including breast, pancreatic, lung, or ovarian cancer (but not limited thereto), provided that the cancer cells exhibit expression of proteins from the group CEACAM1, CEACAM3, CEACAM5, and CEACAM6.

[0056] Example 5

[0057] Analysis of cytotoxicity of 2A3-CAR-T cells against HEK293T lines with overexpression of CEACAM5 and CEACAM6.

[0058] In the HEK293T line, overexpression was generated of individual CEACAM family proteins: CEACAM 1, CEACAM5, and CEACAM6 - in addition to those presented earlier (lines with overexpression of CEACAM 1, CEACAM3, and CEACAM7). Overexpression of the said proteins was tested by Western blot and by flow cytometry (positive staining with antibodies against the respective CEACAM protein, no staining against other proteins from this group). A cytotoxicity test of 2A3-CAR-T cells against the generated lines was conducted by impedance measurement (xCELLinteligence, Agilent). In all lines with overexpression (one clone overexpressing CEACAM1 and two clones overexpressing CEACAM5 and CEACAM6), a significant cytotoxic effect of 2A3-CAR-T cells was found. In the MOCK control line, no cytotoxic effect was observed.

[0059] The data presented confirm that the specificity of the 2A3 antibody, and thus of 2A3- CAR-T cells, depends on whether proteins from the group CEACAM 1, CEACAM3, CEACAM5, and CEACAM6 are present on the surface of the cells. The type of cell line does not affect recognition of epitopes by the 2A3 antibody, because CEACAM5 and CEACAM6 were recognized in both the HEK293T and MDA-MB-231 lines when overexpression of the said proteins was induced in them.

Claims

Claims

1. A 2A3 antibody, characterized in that it exhibits multispecificity toward CEACAM family proteins selected from the group comprising CEACAM 1, CEACAM3, CEACAM5, and CEACAM6.

2. The 2A3 antibody according to claim 1, characterized in that theCEACAM family proteins comprise an epitope containing the amino acid sequence LFGYSWYKG.

3. The 2A3 antibody according to claim 1, characterized in that theCEACAM family proteins comprise an epitope containing the amino acid sequence GYSWYKG.

4. The 2A3 antibody according to any of the preceding claims, characterized in that the antibody is fused to an Fc fragment.

5. A nucleic acid molecule encoding the antibody according to any of the preceding claims.

6. A vector comprising the nucleic acid molecule according to claim5.

7. A construct comprising a CAR with the antibody according to any of claims 1 to 4.

8. The 2A3 antibody according to any of claims 1 to 4 for use in therapy.

9. The 2A3 antibody for use according to claim 8, wherein the therapy is selected from the group comprising cellular immunotherapy, treatment of autoimmune diseases, a therapy in which the 2A3 antibody is conjugated to a cytotoxic drug or a PET tracer, radioimmunotherapy, photoimmunotherapy, a therapy employing a bispecific or trispecific activator of T lymphocytes or natural killer (NK) cells, an immunomodulatory therapy using 2A3 as a blocking antibody or as an antibody with a silenced Fc, a targeted drug-delivery and diagnostic therapy in which 2A3 is linked to liposomes, gold nanoparticles, or polymeric micelles, or is labelled with a dye for intraoperative tumor visualization, or is fused to a viral protein for infection of CEACAM-positive cells.

10. The 2A3 antibody according to any of claims 1 to 4 for use inCAR-T therapy for treating tumors exhibiting overexpression of a protein selected from CEACAM 1, CEACAM3, CEACAM5, CEACAM6, or all of the foregoing simultaneously.8

11. The 2A3 antibody according to any of claims 1 to 4 for use inCAR-T therapy for treating tumors exhibiting overexpression of a protein selected from CEACAM1, CEACAM3, CEACAM5, or all of the foregoing simultaneously.

12. The 2A3 antibody according to any of claims 1 to 4 for use inCAR-T therapy for treating tumors, characterized in that the tumor does not exhibit overexpression of CEACAM6.

13. The 2A3 antibody according to any of claims 1 to 4 for use inCAR-T therapy for treating tumors, characterized in that the tumor is a solid tumor selected from the group comprising cancer of the pancreas, breast, gallbladder and biliary tract, colon, lung, urinary bladder, uterus, prostate, gastrointestinal tract, thyroid, testis, head and neck, and melanoma.

14. The 2A3 antibody according to any of claims 1 to 4 for use inCAR-T therapy for treating tumors, characterized in that the proteins selected from CEACAM1, CEACAM3, and CEACAM5 comprise an epitope containing the amino acid sequence LFGYSWYKG.

15. The 2A3 antibody according to any of claims 1 to 4 for use inCAR-T therapy for treating tumors, characterized in that patient selection includes confirmation of the absence of CEACAM6 overexpression in tumor cells and the overexpression in tumor cells of CEACAM1, CEACAM3, and CEACAM5 proteins comprising an epitope containing the amino acid sequence LFGYSWYKG.9

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